A cyanide-aldehyde complex inhibits bacterial luciferase.

Cyanide at high (millimolar) concentrations inhibited in the in vitro Vibrio harveyi luciferase reaction. Cyanide reacted with free aldehyde to form an inhibitor. Inhibitor formation was accelerated by alkaline conditions and bovine serum albumin.     

Purification and properties of Renilla reniformis luciferase.

Luciferase from the anthozoan coelenterate Renilla reniformis (Renilla luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5.) catalyzes the bioluminescent oxidation of Renilla luciferin producing light (lambdaB 480 nm, QB 5.5%), oxyluciferin, and CO2 (Hori, K., Wampler, J.E., Matthews, J.C., and Cormier, M.J. (1973), Biochemistry 12, 4463). Using a combination of ion-exchange, molecular-sieve, sulfhydryl-exchange, and affinity chromatography, luciferase has been purified, approximately 12 000-fold with 24% recovery, to homogeneity as judged by analysis with disc and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and ultracentrifugation. Renilla luciferase is active as a nearly spherical single polypeptide chain monomer of 3.5 X 10(4) daltons having a specific activity of 1.8 X 10(15) hp s-1 mg-1 and a turnover number of 111 mumol min-1 mumol-1 of enzyme. This enzyme has a high content of aromatic and hydrophobic amino acids such that it has an epsilon280nm 0.1% of 2.1 and an average hydrophobicity of 1200 cal residue-1. The high average hydrophobicity of luciferase, which places it among the more hydrophobic proteins reported, is believed to account, at least in part, for its tendency to self-associate forming inactive dimers and higher molecular weight species.     

Secretion of firefly luciferase in E.coli

Summary A DNA segment carrying the full-length, intronless firefly luciferase gene was inserted into the high expression secretion vector, pIN-III -ompA. Upon induction of gene expression, luciferase activity was detected in extracts prepared from periplasmic fractions. The results indicated that the OmpA signal peptide was able to direct secretion of firefly luciferase across the cytoplasmic membrane. This has important implications for using this luciferase as a reporter in studying protein export and targeting.     

Fluorescence and bioluminescence of bacterial luciferase intermediates.

An intermediate in the luciferase-catalyzed bioluminescent oxidation of FMNH2, isolated and purified by chromatography at -20degrees, was postulated to be an oxygenated reduced flavin-luciferase. Maintained and studied at -20 to -30degrees, this material exhibits a relatively weak fluorescence emission peaking about 505 nm when excited at 370 nm. It may comprise more than one species. Upon continued exposure to light at 370 nm, the intensity of this fluorescence increases, often by a factor of 5 or more, and its emission spectrum is blue shifted to a maximum at about 485 nm. Upon warming its fluorescence is lost and the fluorescence of flaving mononucleotide appears. If warming is carried out in the presence of a long chain aldehyde, bioluminescence occurs, with the appearance of a similar amount of flavine fluorescence. The bioluminescence yield is about the same with irradiated and nonirradiated samples. The bioluminescence emission spectrum corresponds exactly to the fluorescence emission spectrum of the intermediate formed by irradiation, implicating the latter as being structurally close to the emitting species in bioluminescence.     

Immobilized luciferase of Luciola mingrelica fireflies. The kinetic properties and thermostability of luciferase immobilized on cellulose films

Luciferase of fireflies Luciola mingrelica was immobilized on cellulose films activated by cyanuric chloride or sodium periodate. Kinetic properties and the contribution of diffusional obstacles to the kinetics of the immobilized enzyme were examined. External and internal diffusion were found to influence the kinetic parameters. The stability of the enzyme was investigated at 25 degrees C and pH 7.8. Thermoactivation of the immobilized enzyme was shown to proceed in two stages: fast and slow. Dithiotreitol and cystein stabilized the enzyme at the fast stage while salt supplements at both stages. The fast thermoinactivation stage was apparently associated with the oxidation of luciferase SH-groups. It is demonstrated that the immobilized enzyme of Luciola mingrelica can be employed to measure ATP traces with the detection limit 0.1 mM. The enzyme immobilized on cellulose films can be used repeatedly.     

Gaussia luciferase for bioluminescence tumor monitoring in comparison with firefly luciferase

Gaussia luciferase (Gluc) is a secreted reporter, and its expression in living animals can be assessed by in vivo bioluminescence imaging (BLI) or blood assays. We characterized Gluc as an in vivo reporter in comparison with firefly luciferase (Fluc). Mice were inoculated subcutaneously with tumor cells expressing both Fluc and Gluc and underwent Fluc BLI, Gluc BLI, blood assays of Gluc activity, and caliper measurement. In Gluc BLI, the signal from the tumor peaked immediately and then decreased rapidly. In the longitudinal monitoring, all measures indicated an increase in tumor burden early after cell inoculation. However, the increase reached plateaus in Gluc BLI and Fluc BLI despite a continuous increase in the caliper measurement and Gluc blood assay. Significant correlations were found between the measures, and the correlation between the blood signal and caliper volume was especially high. Gluc allows tumor monitoring in mice and should be applicable to dual-reporter assessment in combination with Fluc. The Gluc blood assay appears to provide a reliable indicator of viable tumor burden, and the combination of a blood assay and in vivo BLI using Gluc should be promising for quantifying and localizing the tumors.     

Model of the active site of firefly luciferase.

A model for the spatial structure of firefly luciferase--ATP--luciferin complex is suggested using the coordinates of unliganded luciferase and the enzyme--substrate complex of the adenylating subunit of gramicidin S synthetase known from the literature. Conformational changes in luciferase can occur during substrate binding resulting in a relative orientation of two luciferase domains similar to that in case of the AMP--phenylalanine--synthetase complex. The model is consistent with data on the physicochemical properties of firefly luciferase and its complexes with the substrates.     

Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase.

In the presence of ATP, luciferin (LH2), Mg2+ and pyrophosphatase, the firefly (Photinus pyralis) luciferase synthesizes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) through formation of the E-LH2-AMP complex and transfer of AMP to ATP. The maximum rate of the synthesis is observed at pH 5.7. The Km values for luciferin and ATP are 2-3 microM and 4 mM, respectively. The synthesis is strictly dependent upon luciferin and a divalent metal cation. Mg2+ can be substituted with Zn2+, Co2+ or Mn2+, which are about half as active as Mg2+, as well as with Ni2+, Cd2+ or Ca2+, which, at 5 mM concentration, are 12-20-fold less effective than Mg2+. ATP is the best substrate of the above reaction, but it can be substituted with adenosine 5'-tetraphosphate (p4A), dATP, and GTP, and thus the luciferase synthesizes the corresponding homo-dinucleoside polyphosphates:diadenosine 5',5"'-P1,P5-pentaphosphate (Ap5A), dideoxyadenosine 5',5"'-P1,P4-tetraphosphate (dAp4dA) and diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G). In standard reaction mixtures containing ATP and a different nucleotide (p4A, dATP, adenosine 5'-[alpha,beta-methylene]-triphosphate, (Ap[CH2]pp), (S')-adenosine-5'-[alpha-thio]triphosphate [Sp)ATP[alpha S]) and GTP], luciferase synthesizes, in addition to Ap4A, the corresponding hetero-dinucleoside polyphosphates, Ap5A, adenosine 5',5"'-P1,P4-tetraphosphodeoxyadenosine (Ap4dA), diadenosine 5',5"'-P1,P4-[alpha,beta-methylene] tetraphosphate (Ap[CH2]pppA), (Sp-diadenosine 5',5"'-P1,P4-[alpha-thio]tetraphosphate [Sp)Ap4A[alpha S]) and adenosine-5',5"'-P1,P4-tetraphosphoguanosine (Ap4G), respectively. Adenine nucleotides, with at least a 3-phosphate chain and with an intact alpha-phosphate, are the preferred substrates for the formation of the enzyme-nucleotidyl complex. Nucleotides best accepting AMP from the E-LH2-AMP complex are those which contain at least a 3-phosphate chain and an intact terminal pyrophosphate moiety. ADP or other NDP are poor adenylate acceptors as very little diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) or adenosine-5',5"'-P1,P3-triphosphonucleosides (Ap3N) are formed. In the presence of NTP (excepting ATP), luciferase is able to split Ap4A, transferring the resulting adenylate to NTP, to form hetero-dinucleoside polyphosphates. In the presence of PPi, luciferase is also able to split Ap4A, yielding ATP. The cleavage of Ap4A in the presence of Pi or ADP takes place at a very low rate. The synthesis of dinucleoside polyphosphates, catalyzed by firefly luciferase, is compared with that catalyzed by aminoacyl-tRNA synthetases and Ap4A phosphorylase.     

Enhancement of firefly luciferase activity by cytidine nucleotides.

The temporal pattern of light production by firefly luciferase depends on the ATP concentration. With low concentrations of ATP a constant production of light occurred while at high concentrations of ATP (greater than 10 microM) there was a flash of light followed by a decline in light production. This time course of light production with high ATP concentrations was changed from the flash pattern to a pattern with a constant production of light by several cytidine nucleotides. CTP, CDP, dCTP, dCDP, dideoxyCTP, periodate-oxidized CTP and CDP, and the etheno derivatives of CTP and CDP produced that change. CMP, cytidine, CDP-glycerol, CDP-glucose, CDP-ethanolamine, and benzoylbenzoylCTP either were inhibitory to firefly luciferase or were not effective in changing the flash time course. Coenzyme A and related compounds also changed the time course of light production. The changes in time course produced by either cytidine nucleotides or CoA were inhibited by desulfoCoA. These compounds apparently enhanced light production by promoting the dissociation of the inhibitory product, oxidized luciferin, from the enzyme. When the activating compounds were used with high concentrations of ATP, the sensitivity of assay for firefly luciferase was increased. This increased sensitivity is important when using the firefly luciferase gene as a reporter.     

Stopped-flow kinetic analysis of the bacterial luciferase reaction.

The kinetics of the reaction catalyzed by bacterial luciferase have been measured by stopped-flow spectrophotometry at pH 7 and 25 degrees C. Luciferase catalyzes the formation of visible light, FMN, and a carboxylic acid from FMNH2, O2, and the corresponding aldehyde. The time courses for the formation and decay of the various intermediates have been followed by monitoring the absorbance changes at 380 and 445 nm along with the emission of visible light using n-decanal as the alkyl aldehyde. The synthesis of the 4a-hydroperoxyflavin intermediate (FMNOOH) was monitored at 380 nm after various concentrations of luciferase, O2, and FMNH2 were mixed. The second-order rate constant for the formation of FMNOOH from the luciferase-FMNH2 complex was found to be 2.4 x 10(6) M-1 s-1. In the absence of n-decanal, this complex decays to FMN and H2O2 with a rate constant of 0.10 s-1. The enzyme-FMNH2 complex was found to isomerize prior to reaction with oxygen. The production of visible light reaches a maximum intensity within 1 s and then decays exponentially over the next 10 s. The formation of FMN from the intermediate pseudobase (FMNOH) was monitored at 445 nm. This step of the reaction mechanism was inhibited by high levels of n-decanal which indicated that a dead-end luciferase-FMNOH-decanal could form. The time courses for these optical changes have been incorporated into a comprehensive kinetic model. Estimates for 15 individual rate constants have been obtained for this model by numeric simulations of the various time courses.     

Mechanism of inhibition of bacterial luciferase by anaesthetics.

The effects of volatile anaesthetics on bacterial luciferase were studied $\text{in vitro}$. It was shown that the concentration of anaesthetic required to inhibit the reaction velocity by 50% was similar to that required to reduce light output by 50% $\text{in vivo}$ and this concentration was also in the clinical range for each agent. A kinetic response suggestive of competitive inhibition is occuring at the aldehyde binding site on the luciferase and it is postulated that this is related to the very hydrophobic nature of this site.     

Flavin binding by bacterial luciferase: Affinity chromatography of bacterial luciferase

Immobilized FMN covalently attached to Sepharose-6B-hexanoate binds bacterial luciferase. Immobilized flavin is also effective in its reduced form as a substrate in the light emitting reaction catalyzed by luciferase.