Luciferase activity

Orlova et al. (2003) theoretically studied the mechanism of the firefly bioluminescence reaction on the basis of the hybrid density functional theory. According to their conclusion, changes in the color of light emission by rotating the two rings on the 2-2 axis is unlikely, whereas the participation of the enol-forms of oxyluciferin in bioluminescence is plausible but not essential to explain the multicolor emission. They predicted that the color of the bioluminescence depends on the polarization of the oxyluciferin molecule (at its OH and O-termini) in the microenvironment of the luciferase active site the... 

If the luciferase sample solution contains a flavin-reductase, luciferase activity can be measured by the addition of FMN and NADH, instead of FMNH2. In this case, the turnover of luciferase takes place repeatedly using the FMNH2 that is enzymatically generated thus, the luminescence reaction continues until aldehyde or NADH is exhausted. A crude luciferase extracted from luminous bacteria usually contains a flavin-reductase. 

Shrimps of the genus Sergestes normally emit light from their photophores. However, S. lucens, a species abundantly harvested in Japan as a delicacy Sakura-ebi, has never been observed to emit light despite its specific name and the clear presence of photophores (Haneda, 1985). This species (body weight 0.4 g) undoubtedly contains some coelenterazine, but shows very little luciferase activity, which seems to be insufficient as evidence for the existence of a luciferase (Shimomura et al., 1980 see the note at the end of Section 5.2 for the reason). Thus, the luminescence of S. lucens might involve a coelenterazine luminescence system different from... 

Purification of luciferase. Cypridina luciferase is more stable than many other luciferases, except that this enzyme is rapidly inactivated at acidity below pH 5.0. The dried specimens that have been stored for over 50 years at room temperature (sometimes exceeding 30°C) still possess strong luciferase activity that can be extracted and purified. Preparations of highly purified luciferase have been obtained by various methods (McElroy and Chase, 1951 Shimomura et al., 1961,1969 Tsuji and Sowinski, 1961 Stone, 1968 Tsuji etal., 1974 Thompson et al., 1989) the purification methods employed include... 

Dried shrimp was ground, defatted with benzene, and then extracted with cold water. The luciferase extracted was purified first by a batch adsorption onto DEAE cellulose (elution with 0.4 M NaCl), followed by gel filtration on a column of Sephadex G-150, anion-exchange chromatography on a column of DEAE-cellulose (gradient elution 0.05-0.5 M NaCl), and gel filtration on a column of Ultrogel AcA 34. The specific activity of the purified luciferase was 1.7 x 1015 photons s 1 mg-1, and the yield in terms of luciferase activity was about 28%. 

Quantum yield and luciferase activity The quantum yield of coelenterazine in the luminescence reaction catalyzed by Oplophorus luciferase was 0.34 when measured in 15 mM Tris-HCl buffer, pH 8.3, containing 0.05 M NaCl at 22°C (Shimomura et al., 1978). The specific activity of pure luciferase in the presence of a large excess of coelenterazine (0.9pg/ml) in the same buffer at 23°C was 1.75 x 1015 photons s 1 mg-1 (Shimomura et al., 1978). Based on these data and the molecular weight of luciferase (106,000), the turnover number of luciferase is calculated at 55/min. 

The particulate matter can be solubilized with 2-mercaptoethanol, giving a mixture of luciferase oligomers with molecular masses in multiples of approximately 20kDa. The luciferase activity is increased several times by the solubilization. The purification of the solubilized... 

Fig. 4.5.4 Heat stability of Periphylla luciferases A, B, C and L in 20 mM Tris-HC1 buffer (pH 7.8) containing 1 M NaCl and 0.05% BSA (solid lines) or 0.01% LCC (dotted lines). The buffer (1 ml) containing a luciferase sample was placed in a glass test tube that had been pre-equilibrated at a temperature in a water-bath. After 2 min, the test tube was briefly cooled in cold water, and then luciferase activity in 10 jl1 of the solution was measured in 3 ml of the pH 7.8 buffer containing 0.3 i,M coelenterazine at 24° C. From Shimomura et al., 2001.

Fig. 4.5.5 Effect of pH on the luminescence of coelenterazine catalyzed by Periphylla luciferases A, B and C, and on the stability of the luciferases. The effect on light intensity (solid lines) was measured in 3 ml of 50 mM phosphate buffers, pH 4.1-7.25, and 50 mM Tris-HCl buffers, pH 7.1-9.7, all containing 1 M NaCl, 0.025% BSA, and 0.3 pM coelenterazine. To measure the stability (dotted lines), a luciferase sample (5 pi) was left standing for 30 min at room temperature in 0.1 ml of a buffer solution containing 1 M NaCl and 0.025% BSA and having a pH to be tested, and then luciferase activity in 10 pi of the solution was measured in 3 ml of 20 mM Tris-HCl, pH 7.8, containing 1M NaCl, 0.05% BSA, and 0.3 pM coelenterazine at 24°C. The amounts of luciferases used for measuring each point were luciferase A, 150 LU luciferases B and C, 170 LU. One LU = 5.5 x 108 quanta/s. From Shimomura etal., 2001.

Table 5.1 Contents of Coelenterazine and Luciferase Activity in Individual Specimen of Marine Luminous and Nonluminous Organisms. Luciferase Activities are Shown in the Parentheses in the Last Column...

A note on the assays of coelenterazine and luciferase activity. The methods for measuring coelenterazine and the corresponding luciferases are given in Appendix C5. Special attention must be paid to the fact that coelenterazine in aqueous buffer solutions spontaneously emits a low level of chemiluminescence in the absence of any luciferase, which is greatly enhanced by the presence of various substances, including egg yolk, BSA and various surfactants (especially, hexadecyltrimethylammonium bromide). Therefore, the utmost care must be taken in the detection and measurement of a low level of... 

Luciferase activity on e-coelenterazine. In the presence of Renilla luciferase, the luminescence intensity of e-coelenterazine is more than 5 times higher than that of coelenterazine under the same conditions... 

Luciferase-catalyzed luminescence of luciferin. Odontosyllis luciferin emits light in the presence of Mg2+, molecular oxygen and luciferase. The relationship between the luminescence intensity and the pH of the medium shows a broad optimum (Fig. 7.2.8). The luminescence reaction requires a divalent alkaline earth ion, of which Mg2+ is most effective (optimum concentration 30 mM). Monovalent cations such as Na+, K+, and NH have little effect, and many heavy metal ions, such as Hg2+, Cu2+, Co2+ and Zn2+, are generally inhibitory. The activity of crude preparations of luciferase progressively decreases by repeated dialysis and also by concentrating the solutions under reduced pressure. However, the decreased luciferase activity can be completely restored to the original activity by the addition of 1 mM HCN (added as KCN). The relationship between the concentration of HCN and the luciferase activity is shown in Fig. 7.2.9. Low concentrations of h and K3Fe(CN)6 also enhance luminescence, but their effects are only transient. 

This luminous brittle star has been briefly studied recently (Mallefet and Shimomura, 2004, unpublished). The animal contained a high level of coelenterazine luciferase activity (4 x 1012 photons s-1g 1), which is comparable to those in the luminous antho-zoans such as the sea pansy Renilla and sea pen Ptilosarcus (Shimomura and Johnson, 1979b). There is no evidence for the presence of a photoprotein in this brittle star. Thus, the luminescence system of Amphiura filiformis is considered to be a coelenterazine-luciferase system, differing from that of Ophiopsila californica. The luciferase has a molecular weight of 23,000 on the basis of gel filtration on Superdex 200 Prep, and catalyzes the luminescence reaction of coelenterazine in the presence of oxygen the light emission (A.max 475 nm) is optimum at pH 7.2. 

The two species of Diaphus mentioned above contained relatively large amounts of coelenterazine and also exhibited weak but unmistakable luciferase activities (Shimomura et al., 1980). Thus, it is... 

If a trace activity is indicated by the luminescence intensity measurement, the following two methods can be used to determine whether the light emission is due to the luciferase or it is an artifact (1) Measure the luminescence intensity with a buffer that contains 1 mM EDTA (add luciferase to this buffer and wait 1 min before mixing with luciferin). If the luminescence was caused totally by luciferase, the light intensity will be decreased to about 20% by EDTA (see Section 3.1.7). (2) Inactivate luciferase by acidifying the sample to pH about 2.0, followed by neutralization with NaHCC>3. Inactivated luciferase should not show any luciferase activity. 

Coelenterazine and the corresponding luciferase can be easily tested in the field. A small piece of tissue sample is put in a test tube with methanol (for coelenterazine) or water (for luciferase), and crushed with a spatula. To measure coelenterazine, a buffer solution containing a coelenterazine luciferase is injected into a small amount of the fluid part of the crushed sample mixture. Similarly, luciferase can be measured with a buffer solution containing coelenterazine. The presence of Cypridina luciferin can be tested in the same fashion, with the methanol extract of samples and crude Cypridina luciferase. However, the detection of a very weak Cypridina luciferase activity in the field is not recommended (see Section C5.6). To test the presence of a Ca2+-sensitive photoprotein, crush a sample in a neutral buffer solution containing 20-50 mM EDTA, and then add lOmM calcium acetate to a small portion of the fluid part of the crushed sample to detect any light emission. 

Branchini, B. R., et al. (1997). Identification of a firefly luciferase active site peptide using a benzophenone-based photooxidation reagent. J. Biol. Cbem. 272 19359-19364. 

Dunlap, J. C., and Hastings, J. W. (1981). The biological clock in Gonyaulax controls luciferase activity by regulating turnover. J. Biol. Chem. 256 10509-10518. 

Ford, S. R., Hall, M. S., and Leach, F. R. (1992). Enhancement of firefly luciferase activity by cytidine nucleotides. Anal. Biochem. 204 283-291. 

Rees, J. F., Thompson, E. M., Baguet, F., and Tsuji, F. I. (1990). Detection of coelenterazine and related luciferase activity in the tissues of the luminous fish, Vinciguerria attenuata. Comp. Biochem. Physiol. 96A 425-430. 

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