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At least 37 records · Page 2Linked to original sources
PubMed1979

Evidence for a fatty acid reductase catalyzing the synthesis of aldehydes for the bacterial bioluminescent reaction. Resolution from luciferase and dependence on fatty acids.

The enzyme responsible for the stimulation by ATP AND NADPH of light emission catalyzed by bacterial luciferase has been partially purified from extracts of the luminescent bacterium, Photobacterium phosphoreum. The stimulatory activity was found to be stabilized by high concentrations of mercaptoethanol, permitting it to be separated from luciferase into an active and stable form and enabling further characterization of its functional properties. The activity of the enzyme was shown to be dependent not only on ATP and NADPH but also on the presence of a long chain fatty acid, and was inhibited by the addition of NADH and horse liver alcohol dehydrogenase. The specificity for fatty acids, as measured by the stimulation of luciferase activity, had a very limited range, with maximal luminescence being obtained with myristic acid and lower responses being observed only with tridecanoic and pentadecanoic acid. These results provide evidence in vitro for an enzyme in bioluminescent bacteria that functions as a fatty acid reductase converting fatty acids to aldehydes which in turn can be utilized by luciferase in the light-emitting reaction.

Alcohol Oxidoreductases↗
PubMed1975

Characterization and properties of Pholas luciferase as a metalloglycoprotein.

The luciferase of the bioluminescent boring mollusc, Pholas dactylus, has been purified by a new method which includes centrifugation in cesium chloride gradients. Homogeneous preparations have been obtained and molecular weight determinations and subunit analysis support the idea that this preparation is an oxyluciferin-luciferase complex. The preparation catalyzes oxidation of ascorbic acid in presence of H2O2, and this peroxidase activitity has been used for characterization (thermal and pH stabilities, activity as a function of pH, isoelectric point, turnover number). The existence of two atoms of copper has been established and their involvement in the peroxidase activity indicated. Chemical analyses have shown that Pholad luciferase is a glycoprotein and the existence of glucosamine, fucose, mannose, and galactose residues has been demonstrated. The apparent buoyant dentisty (1.340), the sedimentation coefficient (10.7 S), the Stoke's radius (83 A), the partial specific volum (0.707), and the molecular weight (350,000) have been determined. The frictional ratio (flf0 = 1.8) derived from the Stoke's radius indicates that the molecule is asymmetric. The quaternary structure has been examined. Subunits of molecular weight 150,000 and 46,000 have been observed. The latter has electrophoretic properties identical with luciferin or oxyluciferin.

Amino Acids↗
PubMed1975

Bacterial luciferase requires one reduced flavin for light emission.

Recent reports revive a hypothesis that the bacterial bioluminescence reaction involves two reduced flavin mononucleotide molecules per enzyme turnover. A two-flavin mechanism requires that the two flavins bind simultaneously or sequentially to the same or different sites on luciferase during a catalytic cycle. Measurements using equilibrium techniques show that the luciferase dimer has only a single reduced flavin binding site. Quantum yield results demonstrate that bioluminescence requires only one reduced flavin per luciferase, ruling out mechanisms involving either two reduced flavins or one reduced flavin plus one oxidized flavin per catalytic cycle.

Flavin Mononucleotide↗
PubMed1977

Sequential chemical modification of a histidyl and a cysteinyl residue in bacterial luciferase.

Sequential modification of two amino acid residues (a histidyl and a cysteinyl residue), both essential for the enzymatic function of bacterial luciferase from Beneckea harveyi, has been conducted to determine if the inactivation arising from the chemical modification of either of these residues is due to a conformational change. This experimental approach has shown that modification of the histidyl or cysteinyl residue did not affect the reactivity of the remaining 'essential' residue, suggesting that chemical modification had not caused a change in conformation. Furthermore, since substrates protect luciferase against inactivation due to modification of either of these residues, it was possible to determine if the initial modification of the histidyl or cysteinyl residue prevented substrate binding by conducting the modification of the remaining residue (i.e., the cysteinyl or histidyl residue, respectively) in the presence of substrates. The results have shown that after modification of the histidyl residue substrates no longer protected the cysteinyl residue against modification, whereas after modification of the cysteinyl residue substrates still protected the histidyl residue against modification. These results have provided evidence that the histidyl residue and not the cysteinyl residue of luciferase is essential for the binding of substrates in the bacterial bioluminescent reaction.

Bacteria↗
PubMed1979

[Kinetic properties of highly purified luciferase from fireflies Luciola mingrelica].

Luciferase of the fireflies Luciola mingrelica was isolated from dried lanterns of fireflies and purified by chromatography on DEAE-Sephadex. The homogeneity of the preparation was determined by polyacrylamide gel disc electrophoresis. The molecular weight of the enzyme equal to 45000 was determined by disc electrophoresis in the presence of sodium dodecyl sulfate. The kinetic properties of the enzyme (V and Km for luciferin and ATP) within the pH-range of 7,0--8,5 were studied. The kinetic curves of the pH-dependences of log V and log Km for both substrates are bell-shaped, with a slope equal to 2. At pH optimum (7,7--7,9) the Km values for luciferin and ATP are 6,6 mkM and 0,3 mM, respectively. The properties of luciferase L. m. were compared to those of luciferase from fireflies Phophinus pyralis previously described in literature.

Animals↗
PubMed2026

Development of receptor and oncogene-responsive luciferase reporter vectors that are activated via kinase signaling to AP-1.

This study aimed to generate lentiviral vectors carrying an array of AP-1 motifs driving luciferase gene expression as reporters of mitogen-activated protein kinase (MAPK) activity. We created a series of vectors based on LeGO-iG that were used to generate stably transduced leukemia cell lines. A vector termed LEGO-AP1×6-GM55 containing an array of 6 AP-1 sites linked to the minimal CSF2 promoter was sufficient to support high levels of MAPK-inducible luciferase activity in leukemic cell lines that was suppressed by MAPK inhibitors. The inclusion of a putative chromatin priming element encompassing RUNX and ETS motifs increased the activity of these vectors. The additional inclusion of the full-length mouse CSF2 promoter, or the human DUSP5 promoter further increased the MAPK-dependent activity of these vectors in leukemic cells. These vectors support moderate levels of constitutive activity in cells carrying mutations that activate the RAS/RAF/MEK MAPK signaling pathway, and high-level activity after direct activation of MAPK signaling. They also respond to T-cell receptor activation via MAPK and Ca2+ signaling pathways. This resource will now make it easier to track receptor or oncogene-inducible MAPK activity in cultured cells, and potentially in tumors, close to real-time.

Humans↗
PubMed1978

Plasma ADP levels: direct determination with luciferase luminescence using a biometer.

A method is described for the determination of low plasma levels of adenosine-5'-diphosphate (ADP) using a Dupont Biometer to measure luminescence produced by the luciferin-luciferase reaction. Endogenous ATP is removed by incubation with luciferase. The remaining ADP is then quantitated, following its conversion to ATP, after incubation with creatine phosphate and creatine kinase. The mean coefficient of variation for 0.02 and 2.2 micromol/liter ADP standards were 2.1 and 1.8% respectively. The method has been applied to human and rabbit plasma. Human plasma ADP concentrations were found to be 0.13 +/- 0.025 (10) micromol/liter and rabbit plasma concentration were 0.07 +/- 0.05 (5) micromol/liter. Several other possible applications of the method are discussed.

Adenosine Diphosphate↗
PubMed1979

Interactions of long-chain aldehydes with luciferase. A carbon-13 nuclear magnetic resonance study.

The interaction of long-chain aldehydes with bacterial luciferase has been studied by 13C NMR spectroscopy of natural-abundance and 13C-enriched 1-dodecanal. At high substrate/enzyme ratios, the spin-spin relaxation rates of C(1)-C(3) are faster than for the other carbons and are in the order C(1) greater than C(2) greater than C(3). The aldehyde is strongly bound in the active site along the entire length of the alkyl chain with the strongest interaction at the CHO group. At low substrate/enzyme ratios, interactions are apparent at C(10), which are removed upon denaturation of the enzyme. Spin-spin and spin-lattice relaxation rates were measured for odd-carbon 13C-enriched 1-dodecanal in the presence of luciferase. From the ratios of T1/T2 a single value of (1.8 +/- 0.7) X 10(-8) s was calculated for the rotational correlation time tc for the complex.

Aldehydes↗
PubMed1977

Kinetics of product inhibition during firefly luciferase luminescence.

A theoretical and experimental analysis is made of the kinetics of product inhibition during firefly luciferase luminescence. Equations for competitive, noncompetitive, and uncompetitive inhibition are derived which are useful in determining inhibitory mechanism when the product inhibitor, or its concentration, is unknown and not subject to direct experimental manipulation. Comparisons of experimental data with predictions based upon the three inhibitory models show that product inhibition during luciferase luminescence is noncompetitive with respect to both luciferin and ATP as substrates. The competitive and uncompetitive models are inconsistent with experimental data. These findings provide the basis for using luminescence to measure ATP concentration continuously in in vitro biological systems such as isolated mitochondria.

Animals↗
PubMed1978

Studies on luciferase from Photobacterium phosphoreum. X. Heat of formation of the intermediate in the bioluminescent reaction studied by stopped-flow calorimetry.

The heat production in the reaction of luciferase-FMNH2 complex with O2 in the absence of aldehyde was measured by stopped-flow calorimetry. deltaH of the reaction, luciferase-FMNH2+ O2 leads to intermediate X1, is -1.3 X 10(2) kJ.mol-1 and the calculated deltaS for the reaction is -180 J.mol-1.K-1 at 20 degrees C. The heat production in the bioluminescent reaction was also measured in the presence of a saturating concentration of aldehyde, and it was estimated that 43 and 79% of the C10 and C13 aldehydes, respectively, bound with the intermediate X1 are converted to carboxylic acid yielding energy for photon emission.

Calorimetry↗
PubMed1975

Detection of bacteriuria by luciferase assay of adenosine triphosphate.

A selective method for distinguishing bacterial and nonbacterial adenosine triphosphate (ATP) in clinical bacteriological specimens was studied. The method involved incubation of samples with the detergent Triton X-100 and the ATP-hydrolyzing enzyme apyrase. The incubation selectively destroyed ATP in suspensions of various human cells while not affecting the ATP content in microbial cells. ATP remaining in the sample after incubation was extracted in boiling buffer and assayed by the firefly luciferase assay. Application of the method to 469 clinical urine specimens showed that the ATP level after treatment with Triton/apyrase was correlated to bacterial counts and that the sensitivity of the assay was sufficient for the detection of 10(5) bacteria/ml. The ATP levels per bacterial cell remaining in the urine specimen after treatment with Triton/apyrase were close to values observed in laboratory-grown cultures. The specificity and sensitivity of the luciferase assay for the detection of urinary bacteria and its possible use as a bacteriuria screening method are discussed.

Adenosine Triphosphate↗
PubMed1976

Effect of various ions on ATP determinations using the "luciferine-luciferase" system.

Various salts and buffers used in routine as part of the ATP extraction procedues induce an important inhibition of the peak light emission produced by the "luciferine-luciferase" system. The nature of the anion is more important in determining the inhibitory effect than the nature of the cation. The series obtained when placing the anions studied by order of increasing effectiveness is as follows Ac- less than Cl- less than I- less than ClO4-. KClO4 appears thus as a strong inhibitor of the enzyme activity. It appears moreover to act competitively with respect to ATP, one mole of inhibitor binding per mole of ATP active site. These results are discussed in connection with the use of the "luciferine-luciferase" system for ATP and other energy-rich compounds' concentration measurements.

Adenosine Triphosphate↗
PubMed2025

Use of Rift Valley Fever Virus Expressing NanoLuc Luciferase for the Assessment of Neutralizing Antibodies and Antivirals.

Rift Valley fever (RVF) is an arboviral zoonotic disease affecting many African countries with the potential to spread to other geographical areas. In this chapter we describe the use of a replication-competent recombinant (r)RVFV expressing NanoLuc Luciferase (Nluc) for in vitro studies. The determination of parameters such as neutralizing antibodies in serum samples, or the antiviral activity of drugs is usually carried out using standard assays based on the assessment of cytopathic effect on cell cultures. The use of a virus encoding a traceable reporter protein allows to correlate the presence or absence of infection with the detection of the product in the infected cultures, thus tracking the level of RVFV infection in an objective, quantitative manner. In addition to this quantitative measurement of results, our protocol offers two other advantages, such as a shorter time to read, given that 48 h post-infection the production of the reporter protein is enough to give an accurate result, and the use of an attenuated virus, which reduces the risk of exposure.

Rift Valley fever virus↗
PubMed1977

Low oxygen is optimal for luciferase synthesis in some bacteria. Ecological implications.

The synthesis of the bioluminescent systems in many strains of two species of the genus Photobacterium which were isolated as symbionts is greater at low oxygen concentrations, where aerobic growth is blocked. In strains of two other species, one Photobacterium of symbiotic orgin, and one (genus Beneckea) whose lent response is observed. At low oxygen concentrations, where there is an inhibition of growth, there is also a similar decrease in the synthesis of the luminescent system. These species-specific differences may indicate important ecological differences along with distinctive differences in the molecular control mechanisms involved in the synthesis of luciferase.

Anaerobiosis↗
PubMed1978

Sensitive assay of creatine kinase isoenzymes in human serum using M subunit inhibiting antibody and firefly luciferase.

A sensitive method for the assay of B subunit and total creatine kinase activity is described. The ATP formation in the creatine kinase reaction is continuously monitored by measuring the bioluminescence obtained with a purified firefly luciferase reagent. The B subunit activity is determined using an M subunit inhibiting antibody resulting in greater than 99.5% and approximately 50% inhibition of MM and MB isoenzymes, respectively. The bioluminescent method correlated well with a similar spectrophotometric method in the assay of B subunit as well as total creatine kinase activity (r greater than or equal to 0.98). However, the bioluminescent assay is considerably more sensitive, allowing the assay of B subunit activity in serum from healthy individuals. This is due to the inherent sensitivity of the bioluminescent assay of ATP, a reduced analytical interference from adenylate kinase and a reduced reagent blank. The within-series precision at 1 U/liter and greater than 52 U/liter corresponded to a C.V. of 14% and 3%, respectively. The method is as rapid and suitable for routine work as spectrophotometric methods. From a clinical point of view the new method is of particular interest in the early diagnosis of small acute myocardial infarctions.

Adenylate Kinase↗
PubMed1979

Photokinetic microassay of adenylate kinase using the firefly luciferase reaction.

A new rapid photokinetic method is described for determining the activity of adenylate kinase (ATP:AMP phosphotranspherase, EC 2.7.4.3) in 0.1--5.0 micrograms of freeze-dried tissue. This represents a weight range far below that obtainable by fine-needle biopsy. The reaction 2 ADP in equilibrium with AMP + ATP was employed and the ATP formed assayed with firefly luciferase as light yielder. The light emission was recorded on a multi-channel scaler. The adenylate kinase activities found in tissues of mice were in the same range as previously described in a study using fluorometric microassay.

Adenylate Kinase↗
PubMed1978

Activity and stability of the luciferase--flavin intermediate.

A luciferase intermediate in the bacterial bioluminescence system, which is formed by reaction of enzyme with reduced flavin mononucleotide (FMNH2) and oxygen, is shown to emit light with added aldehyde under anaerobic conditions. The reaction with oxygen is thus effectively irreversible under the conditions used. The flavin chromophore has an absorption maximum at about 370 nm and the potential activity (bioluminescence yield) in the further reaction of the isolated intermediate with aldehyde is strictly proportional to the amount of this flavin chromophore.

Anaerobiosis↗