Luciferase Cypridina

Fig. 1.14 Relationship between the incorporation of 180 into product CO2 and the amount of luciferin used, in the bioluminescence reaction of Cypridina luciferin catalyzed by Cypridina luciferase. The reactions were carried out in H2160 medium with 1802 gas (solid line) in H2lsO medium with 16C>2 gas (dashed line) and the control experiment of the latter using C16C>2 gas instead of luciferin and luciferase (dotted line), all in 20 mM glycylglycine buffer, pH 7.8, containing 40 mM NaCl. From Shimomura and Johnson, 1973a. Reproduced with permission from Elsevier.

It should be noted that Cypridina luciferin emits a fairly strong chemiluminescence in aqueous solutions in the presence of various lipids and surfactants, even in the complete absence of luciferase. The luminescence is especially conspicuous with cationic surfactants (such as hexadecyltrimethylammonium bromide) and certain emulsion materials (such as egg yolk and mayonnaise). Certain metal ions (especially Fe2+) and peroxides can also cause luminescence of the luciferin. Therefore, great care must be taken in the detection of Cypridina luciferase in biological samples with Cypridina luciferin. 

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... 

Inhibitors. Many common enzyme inhibitors show little or no effect on the activity of Cypridina luciferase in the luminescence reaction (Tsuji et al., 1974). However, EDTA strongly inhibits the bioluminescence reaction, showing a peculiar relationship between the... 

Reaction rate. The luminescence reaction of Cypridina luciferin catalyzed by Cypridina luciferase normally follows the first-order... 

Fig. 3.1.4 Bioluminescence spectrum of Cypridina luciferin catalyzed by Cypridina luciferase (A), the fluorescence excitation spectrum of oxyluciferin in the presence of luciferase (B), the fluorescence emission spectrum of the same solution as B (C), and the absorption spectrum of oxyluciferin (D). The fluorescence of oxyluciferin alone and luciferase alone are negligibly weak. Measurement conditions A, luciferin (lpg/ml) plus a trace amount of luciferase in 20 mM sodium phosphate buffer, pH 7.2, containing 0.2 M NaCl B and C, oxyluciferin (20 pM) plus luciferase (0.2mg/ml) in 20 mM sodium phosphate buffer, pH 7.2, containing 0.2 M NaCl D, oxyluciferin (41 pM) in 20 mM Tris-HCl buffer, pH 7.6, containing 0.2 M NaCl. All are at 20°C.

Fig. 3.1.5 Effects of salt concentration on the activity of Cypridina luciferase (solid lines) and quantum yield (dotted lines). In the activity measurement, Cypridina luciferin (1 pg/ml) was luminesced with a trace amount of luciferase in 2.5 mM HEPES buffer, pH 7.5, containing a salt to be tested, at 20°C. In the measurement of quantum yield, luciferin (1 pg/ml) was luminesced with luciferase (20 pg/ml) in 20 mM sodium phosphate buffer (for the NaCl data) or MES buffer (for the CaCl2 data), pH 6.7.

Fig. 3.1.7 Effects of temperature on the activity of Cypridina luciferase (solid line) and the quantum yield of Cypridina luciferin (dashed line). Luciferin (1 pg/ml) was luminesced in the presence of luciferase (a trace amount for the activity measurement 20 pg/ml for the quantum yield) in 50 mM sodium phosphate buffer, pH 6.8, containing 0.1 M NaCl.

The amount of Cypridina luciferin is measured with Cypridina luciferase. To measure the content of Cypridina luciferin in a tissue, the sample is extracted with methanol in the same manner as in the case of coelenterazine (Section C5.1). However, the extracted Cypridina luciferin is extremely unstable in air. Therefore, it is necessary to... 

Cypridina luciferase is not available commercially at present. However, Cypridina luciferase can be readily extracted from both live and dried Cypridina, and the crude extract, after dialysis, can be used in the measurement of Cypridina luciferin. Live Cypridina can be collected in Japan, and the ostracod can be cultivated in laboratory (see the last part of Section 3.1.2). Dried Cypridina is available from certain sources, including the author s laboratory. 

The amount of Cypridina luciferase is measured with Cypridina luciferin. However, the detection and measurement of a trace amount of the luciferase is extremely difficult, for the reason explained below. 

To assay Cypridina luciferase, 1ml of 10 mM pH 7.0 phosphate buffer containing 0.1M NaCl and 5-10 pi of an aqueous... 

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. 

Chase, A. M., and Langridge, R. (1960). The sedimentation constant and molecular weight of Cypridina luciferase. Arch. Biochem. Biophys. 88 294-297. 

Shimomura, O., and Johnson, F. FI. (1975a). Influence of buffer system and pH on the amount of oxygen exchanged between solvent F420 and the C02 produced in the aerobic oxidation of Cypridina luciferin catalyzed by Cypridina luciferase. Anal. Biochem. 64 601-605. 

Tsuji, F. I., and Sowinski, R. (1961). Purification and molecular weight of Cypridina luciferase. J. Cell. Comp. Physiol. 58 125-130. 

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... 

Cypridina luciferase, 62-64, 343 cloning, 63 inhibitors, 63 molecular weight, 63 properties, 63, 64 purification, 62 turnover rate, 68... 

Cypridina luciferin, in the presence of oxygen and the enzyme Cypridina luciferase, is oxidized with emission of light to give products called oxyluciferin and etioluciferin. On the basis of PMR and mass spectra the structure 21 was assigned to oxyluciferin. This is now known to be incorrect. Work on simple analogues of luciferin in aprotic solvents showed that oxidation of compounds such as 22 and 11 was accompanied... 

Renilla and Cypridina luciferases (E.C. 1.13.12.5 - Renilla luciferin oxygen 2-oxidoreductase (decarboxylating) and E.C.. Z. 2.6 Cypri-dina luciferin oxygen 2-oxidoreductase (decarboxylating). 

The reversible heat inactivation of Cypridina luciferase has been demonstrated in a quantitative study by Chase (44a) using highly purified luciferin and partially purified luciferase. 

Reporter DNA stock solution Prepare 1 mL of a DNA reporter stock solution by combining 300 pL of p53-Firefly luciferase (FL), 300 pL of SXTCF-Renilla luciferase (RL), 300 pL of Elkl-Gal4, 60 pL UAS-Cypridina luciferase (GL), and 40 pL of water to achieve a flnal 5 5 5 1 ratio of reporters. The final total DNA concentration of this stock solution is 0.96 mg/ mL. 

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