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)

Two enzymes of ketone body metabolism, 3-oxoacid CoA-transferase and 3-hydroxybutyrate dehydrogenase were measured with ultramicromethods to map their activities along single, dissected segments of mouse nephron. 3-Oxoacid CoA-transferase activity was assayed with a radiochemical procedure by separating [14C]succinyl-CoA, formed in the presence of acetoacetyl-CoA from [1,4-14C]succinate. This procedure, when compared to the spectrophotometric method, resulted in similar activities in mouse organ homogenates. 3-Hydroxybutyrate dehydrogenase activity was determined with sufficient sensitivity by using NADH-dependent luciferase. The dehydrogenase exhibited only 5% of the activity of the 3-oxoacid CoA-transferase when measured in mouse kidney cortex homogenates. Both enzymes were found to be present in all nephron structures studies. The specific activity related to tubular protein, which was determined in parallel in all segments dissected, exhibited a typical distribution pattern along the nephron. Both enzymes roughly paralleled each other along the structures of the distal nephron. They exhibited high activities in the thick ascending limb of Henle's loop and the distal convoluted tubule, but decreased to nearly 20% in the segments of the collecting tubule. In the proximal convoluted and straight tubule 3-oxoacid CoA-transferase activity was almost equal. In contrast, 3-hydroxybutyrate dehydrogenase increased by a factor of 5 from the convoluted to the straight portion. This heterogeneity along the proximal tubule remained when pure D-hydroxybutyrate was used as substrate, but was not found when rat or rabbit nephron segments were analysed. Lowest activities of enzymes were recovered from glomeruli and thin descending limbs of Henle's loop. The results obtained allow for the conclusion that 3-hydroxybutyrate and acetoacetate can be metabolized in all structures of the mouse nephron with different capacities in various segments. With the exception of the relatively low activity of 3-hydroxybutyrate dehydrogenase in the proximal convoluted tubule, the distribution pattern mirrors the distribution of mitochondria along the rat nephron. The results point to the possible role of ketone bodies as energy fuels for nephron segments performing active transport processes.
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PMID:Renal ketone body metabolism. Distribution of 3-oxoacid CoA-transferase and 3-hydroxybutyrate dehydrogenase along the mouse nephron. 658 55

Two different systems of multienzymes have been coimmobilized onto Sepharose and shown to be catalytically active. The first system consists of the five enzymes that catalyze the conversion of glucose 1-phosphate to 1,3-diphosphoglycerate along with the bacterial enzymes which are used to monitor NADH production by light emission. The NADH:FMN oxidoreductase-luciferase enzymes act to "pull" the reactions by continually oxidizing the NADH formed. The second system consisted of the 11 enzymes necessary for the conversion of glucose to alcohol and the bacterial enzymes. In the presence of excess glucose this immobilized system was able to produce 4068 nmol of alcohol per gram of Sepharose-enzymes in 4 h. Using the same system, if 3-phosphoglycerate was used as the substrate, 6552 nmol of alcohol were formed. These multienzyme immobilized systems are models that are uniquely suited for comparative studies of metabolic processes that occur in intact cells.
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PMID:Coimmobilized multienzymes: an in vitro model for cellular processes. 663 54

L(+)-Lactic acid (5 pmol) and D(-)-lactic acid (20 pmol) were assayed by coupling the generation of NADH with the use of bacterial luciferase. The binding of NADH to L(+)-lactic dehydrogenase made it necessary to denature the protein so that the assay with bacterial luciferase was effective. The coupled luciferase assay of L(+)-lactic acid was 400 times more sensitive than the fluorometric assay. The luciferase coupled assay was used to analyze the L(+)- and D(-)-lactic acid contents of small samples of dental plaque.
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PMID:The analysis of picomole amounts of L(+)- and D(-)-lactic acid in samples of dental plaque using bacterial luciferase. 672 Nov 48

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

The effects of NADH:FMN oxidoreductase and luciferase concentrations on the light kinetics of the bacterial bioluminescent reaction were investigated. Light emission with low decay rates was obtained by regulating the conversion of NADH to NAD+ by controlling oxidoreductase activity. Constant light emission can be obtained when the oxidoreductase activity is below 2.5 U/1 in the assay system. The luciferase concentration affects the light intensity but it has no effect on the decay rate of light emission. The substrate decanal and the end-products NAD+ and capric acid had no effect on the light kinetics. The Michaelis constants of bacterial luciferase for FMNH2 and decanal were 3 X 10(-6) M and 8 X 10(-7) M, respectively, and those of oxidoreductase for FMN and NADH were 6.1 X 10(-6) M and 1.6 X 10(-5) M, respectively.
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PMID:The effect of luciferase and NADH:FMN oxidoreductase concentrations on the light kinetics of bacterial bioluminescence. 683 May 92

An automated flow system for the bioluminescent assay of various metabolites have been developed. The enzymes used in the assays have been coimmobilized onto Sepharose and packed into small flow cells. Assays for NADH, glucose 6-phosphate, and primary bile acids utilize the bacterial NADH:FMN oxidoreductase/luciferase and either glucose-6-phosphate dehydrogenase or 7 alpha-hydroxysteroid dehydrogenase. ATP assays were performed using immobilized firefly luciferase. In general, the lower limit of detection of the metabolites was at the picomole level, and light intensity was proportional to the substrate concentration from several picomoles to several hundred picomoles. The reproducibility was good with coefficient of variations in the range of 2-5%. The carryover was less than 5% and 30 samples per hour could be assayed. The flow cells were reusable for up to 700 consecutive assays. The major factor limiting their continued use was bacterial contamination of the Sepharose. The results obtained for serum primary bile acids using the bioluminescent assay wer in good agreement with independent measurements on the same samples using gas-liquid chromatography. The immobilized firefly luciferase system was successfully used to measure high levels of bacteria in urine specimens.
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PMID:Automated bioluminescent assays for NADH, glucose 6-phosphate, primary bile acids, and ATP. 684 38

A simple, rapid, and sensitive bioluminescence method for measuring primary bile acids has been developed and validated. The method is based on enzymatic dehydrogenation of bile acids using a bacterial 7 alpha-hydroxysteroid dehydrogenase that is co-immobilized on Sepharose 4B beads with NADH:FMN oxidoreductase and a bacterial luciferase. The assay is specific for 7 alpha-hydroxy bile acids and has a detection limit of 0.5 pmol/tube, with a linear range of 0.5-50 pmol/tube. The assay shows good precision (6-8% intra-assay; 8-10% inter-assay). The values obtained with the bioluminescence assay showed good agreement with those obtained by gas-liquid chromatography, radioimmunoassay, or endpoint enzymatic assays. When applied to the measurement of serum bile acids, there was no interference from serum albumin, and the effect of other dehydrogenase activity in serum could be eliminated by heating the sample prior to assay. Since the method is rapid (1 minute), extremely sensitive (requires only 10 microliters of serum), and specific, it appears to be the best method currently available for the measurement of serum primary bile acids.
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PMID:Bioluminescence measurement of primary bile acids using immobilized 7 alpha-hydroxysteroid dehydrogenase: application to serum bile acids. 696 70

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

A bioluminescent assay for determination of glycerol using bacterial NADH-linked luciferase was developed and successfully applied to measurement of glycerol release from human fat cells. The procedure is based on enzymic conversion of glycerol to 3-phosphoglycerate, which is irreversible in the presence of arsenate, and subsequent determination of NADH formed in the glycerol-phosphate- and glyceraldehyde-3-phosphate dehydrogenase reactions respectively. Bioluminescent determination of glycerol is about 100 times more sensitive than conventional spectrophotometry, thus permitting more than 100 determinations to be carried out on needle biopsy specimens.
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PMID:Microdetermination of glycerol using bacterial NADH-linked luciferase. 707 66


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