Obelin

A cDNA encoding apoobelin was obtained from O. longissima and sequenced (Illarionov et al., 1995). The deduced amino acid sequence of the apoobelin consists of 195 amino acid residues, with a calculated molecular mass of about 22.2 kDa, closely matching the apoproteins of other Ca2+-sensitive photoproteins such as aequorin from the jellyfish Aequorea (Inouye et al., 1985 Prasher et al., 1985) and clytin from the jellyfish Phialidium gregarium (Inouye and Tsuji, 1993). To obtain recombinant apoobelin, the cDNA encoding apoobelin was expressed in E. coli (Illarionov et al., 2000). The recombinant apoobelin produced was purified and converted into obelin by incubation with coelenterazine in the presence of molecular oxygen and 2-mercaptoethanol or dithioerythritol, as in the case of aequorin. 

Fig. 4.2.1 Luminescence spectra of the Ca2+-triggered light emission of recombinant obelins (dotted lines), and the fluorescence emission spectra of their spent solution after luminescence (solid lines). Left obelin derived from O. geniculata right obelin derived from O. longissima. Reproduced from Markova etal., 2002, with permission from the American Chemical Society.

Concerning the Ca2+-triggered luminescence of obelin, Deng et al. (2001) reported an interesting observation (Fig. 4.2.2) the luminescence of the recombinant obelin from O. longissima is blue (7max 475 nm), whereas a mutant of this obelin, in which the amino acid residue-92 has been changed from tryptophan to phenylalanine, emits... 

Fig. 4.2.2 Left panel-. Uncorrected Ca2+-triggered bioluminescence spectrum of W92F obelin derived from O. longissima. Right panel Corrected bioluminescence spectrum of the same obelin (dotted line), and the fluorescence emission spectrum of the spent solution after luminescence (solid line). From Deng et al., 2001, with permission of the Federation of the European Biochemical Societies.

Deng et al. (2004a,b) prepared the crystals of the spent obelin (W92F mutant from O. longissima) that had been luminesced with Ca2+, and successfully obtained the X-ray structure of apoobelin as an important information in elucidating the mechanism of the luminescence reaction. 

Elimination of a cofactor needed for luminescence. The chelators EDTA and EGTA efficiently quench the luminescence of Ca2+-activated systems such as aequorin, obelin and mnemiopsin. The luminescence systems that require ferrous ions, such as extracts of the polychaete Chaetopterus, can be inhibited by 8-hydroxyquinoline and... 

Minute amounts of coelenterazine can also be measured utilizing apoaequorin or apoobelin (Campbell and Herring, 1990 Thompson et ah, 1995). In this method, a sample containing coelenterazine is treated with an excess amount of apophotoprotein (apoaequorin or apoobelin) to convert it to a Ca2+-sensitive photoprotein (aequorin or obelin). The photoprotein formed is assayed by luminescing it with Ca2+ to determine the amount of coelenterazine originally existed. With this method, the luminescence reaction is fast and usually complete in a few seconds, in contrast to the slower luminescence reactions with luciferases that sometimes require a few minutes to complete. However, the formation of photoprotein from apoaequorin is slow and not necessarily quantitative, and the overall accuracy of the photoprotein method does not compare favorably with that of the luciferase method that directly measures coelenterazine. The author recommends using a luciferase if the enzyme is available. 

Bondar, V. S., etal. (1995). Cadmium-induced luminescence of recombinant photoprotein obelin. Biochim. Biophys. Acta 1231 29-32. 

Campbell, A. K. (1974). Extraction, partial purification and properties of obelin, the calcium-activated luminescent protein from the hydroid Obelia geniculata. Biochem. J. 143 411—418. 

Campbell, A. K., Patel, A. K., Razavi, Z. S., and McCapra, F. (1988). Formation of the calcium-activated photoprotein obelin from apo-obelin and mRNA inside human neutrophils. Biochem. J. 252 143-149. 

Deng, L., et al. (2001). Structural basis for the emission of violet bioluminescence from a W92F obelin mutant. FEBS Lett. 506 281-285. 

Deng, L., et al. (2004a). Preparation and X-ray crystallographic analysis of the Ca2+ -discharged photoprotein obelin. Acta Crystallogr. Ser. D Biol. Crystallogr. 60 512-514. 

Gitelzon, G. I., Tugai, V. A., and Zakharchenko, A. N. (1990). Production of obelin, a calcium-activated photoprotein, from Obelia longissima and its application for registration of the calcium efflux from the fragmented sarcoplasmic reticulum of skeletal muscles. Ukr. Biokhim. Zh. 62 69-76. 

Illarionov, B. A., et al. (1992). Cloning and expression of cDNA coding for the calcium-activated photoprotein obelin from the hydroid polyp Obelia longissima. Dokl. Akad. Nauk SSSR 326 911-913. 

Illarionova, V. A., et al. (1997). Removal of essential ligand in N-terminal calcium-binding domain of obelin does not inactivate the photoprotein or reduce its calcium sensitivity, but dramatically alters the kinetics of the luminescent reaction. In Hastings, J. W., et al. (eds.), Biolu-min. Chemilumin., Proc. Int. Symp., 9th, 1996, pp. 431 —434. Wiley, Chichester, UK. 

Liu, Z.-J., et al. (2003). Atomic resolution structure of obelin soaking with calcium enhances electron density of the second oxygen atom substituted at the C2-position of coelenterazine. Biochem. Biophys. Res. Commun. 311 433-439. 

Markova, S. V., et al. (2002). Obelin from the bioluminescent marine hydroid Obelia geniculata cloning, expression, and comparison of some properties with those of other Ca2+-regulated photoproteins. Biochemistry 41 2227-2236. 

Matveev, S. V., Lewis, J. C., and Daunert, S. (1999). Genetically engineered obelin as a bioluminescent label in an assay for a peptide. Anal. Biochem. 270 69-74. 

Stephenson, D. G., and Sutherland, P. J. (1981). Studies on the luminescent response of the Ca2+-activated photoprotein, obelin. Biochim. Biophys. Acta 678 65-75. 

Vysotski, E. S., Bondar, V. S., and Letunov, V. N. (1989). Extraction and purification of obelin, a calcium-activated photoprotein from the hydroid polyp Obelia longissima. Biokhimiya 54 965-973. 

Vysotskii, E. S., et al. (1990). Extraction, some properties and application of obelin, calcium-activated photoprotein. In Jezowska-Trzebiatowska, B. (ed.), Biol. Lumin., Proc. Int. Sch., 1st, 1989, pp. 386-395. World Scientific, Singapore. 

Vysotski, E. S., etal. (1999). Preparation and preliminary study of crystals of the recombinant calcium-regulated photoprotein obelin from the biolu-minescent hydroid Obelia longissima. Acta Cryst. D55 1965-1966. 

Harmotboe (polynoidin), 243-246 Luminodesmus, 308-314 Obelia (obelins), 133-137 Ophiopsila, 302-306 Phialidium, 137,138 Pholas (pholasin), 194-199 radiolarian, 248 Symplectoteuthis, 206-215 Photoproteins, 346-348 definition, xxi, xxii Photostomias, 338 Pbotostylus, 338 Pboturis, 2, 9, 337 Pbrixothrix, 24, 337 luminescence in two different colors, 24... 

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