Coelenterazine Luciferases

There are many kinds of luminous organisms that utilize coelenterazine as their luciferin. These organisms possess luciferases to catalyze the luminescent oxidation of coelenterazine. Coelenterazine luciferases have been isolated from about 10 kinds of organisms, including the anthozoans Renilla and Ptilosarcus, the scyphozoan 

All of the luciferases cause the emission of a bluish light when they catalyze the oxidation of coelenterazine. However, there are some marked differences between the decapod shrimp luciferases and the cnidarian luciferases (Matthews et al., 1977a,b). For example, the luminescence caused by the former (Amax about 452 nm) is bluer than that caused by the latter (7max 470-480 nm), and the optimum pH of the former, about 8.5, is significantly higher than that of the latter (Renilla, 7.4 Ptilosarcus, 7.0). The optimum temperature of the decapod shrimp luciferases (35°C) is higher than those of Ptilosarcus (23°C) and Renilla (32°C). 

The maximum specific activities of coelenterazine luciferases appear to be in a range of 1015-1016 photons s 1 mg-1, but much 

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 

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The bioluminescence reaction of Oplophorus is a typical luciferin-luciferase reaction that requires only three components luciferin (coelenterazine), luciferase and molecular oxygen. The luminescence spectrum shows a peak at about 454nm (Fig. 3.3.1). The luminescence is significantly affected by pH, salt concentration, and temperature. A certain level of ionic strength (salt) is necessary for the activity of the luciferase. In the case of NaCl, at least 0.05-0.1 M of the salt is needed for a moderate rate of light emission, and about 0.5 M for the maximum light intensity. 

Luminescence activity. The specific luminescence activities (quanta/s emitted from 1ml of a solution of A280nm,icm 1.0) of luciferases A, B and C are in a range of 1.2 4.1 x 1016 photons/s when measured with the standard assay buffer (20 mM Tris-HCl, pFl 7.8, containing 1M NaCl, 0.05% BSA, and 0.14 xg/ml of coelenterazine, at 24°C). These are the highest specific activities of coelenterazine luciferases. 

Quantum yield of coelenterazine. The quantum yields of coelenterazine in the luminescence reaction catalyzed by luciferases A, B and C are all close to 0.30 at 24°C, which is one of the highest values among coelenterazine luciferases. The amount of luciferase L obtained was insufficient to measure reliable data of specific activity and quantum yield. 

Coelenterazine emits chemiluminescence when dissolved in dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) containing a trace amount of base. It also emits bioluminescence in aqueous media in the presence of a coelenterazine luciferase, such as Renilla luciferase or Oplophorus luciferase. In both cases, the luminescence reactions require molecular oxygen. The capability of coelenterazine to produce luminescence is attributed to the presence of the imida-zopyrazinone structure in the molecule. 

Inouye and Shimomura, 1997). With Ptilosarcus luciferase, the luminescence intensity of e-coelenterazine is also significantly higher than that of coelenterazine. With other coelenterazine luciferases, however, the luminescence intensity of e-coelenterazine is generally lower than that of coelenterazine for example, the luminescence intensities of e-coeienterazine measured with the luciferases of the decapod shrimps, the jellyfish Periphylla, and the copepod Pleuromamma, were 50%, 4%, and 0.8%, respectively, in comparison with that of coelenterazine. Thus, the luminescence of coelenterazine catalyzed by Pleuromamma luciferase is suppressed by the addition of e-coelenterazine. 

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 first step of a chemical study should be the quantitative measurements of coelenterazine, dehydrocoelenterazine, and a coelenter-azine-specific luciferase, in the light organs, liver, digestive tract (with empty stomach), and eggs if available (see Section C5 of Appendix for the method). A clear, unequivocal presence of a coelenterazine luciferase indicates the involvement of a luciferin-luciferase system,... 

Coelenterazine can be detected and measured with a coelenterazine luciferase, i.e. a luciferase specific to coelenterazine. As the coelenterazine luciferase, the luciferases from the sea pansy Renilla and the copepods Gaussia and Pleuromamma are commercially available. Certain kinds of decapod shrimps, such as Oplophoms and Heterocarpus, contain a large amount of luciferase, and the luciferases purified from them are most satisfactory for the assay of coelenterazine considering their high activities and high quantum yields. Even partially purified preparations of these luciferases are satisfactory for most measurements. The author routinely uses purified Oplophoms luciferase. 

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. 

Ca2+-sensitive photoproteins, 367 coelenterazine, 362 coelenterazine enol-sulfate, 364 coelenterazine luciferase, 363 Cypridina luciferase, 366 Cypridina luciferin, 365 dehydrocoelenterazine, 365 stabilized coelenterazine, 364 Asteroidea, 231 Astronestbes, 338 Atolla, 91,140, 334 ATP, 3-5,10-16, 23-29 Aracbnocampa luminescence, 26 assay of firefly luciferase, 11 firefly bioluminescence, 3-5, 10-16... 

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