Reference: Biol. Bull. 194: 244-252. (June. 1998) Luciferase of the Scyphozoan Medusa Periphylla periphylla OSAMU SHIMOMURA' * AND PER R. FLOOD-' Marine Biological Laboratory, Woods Hole. Massachusetts 02543; Department of Physiology. Boston University School of Medicine. Boston, Massachusetts 02118: and ' Bathybiologica A.S., N-5030 Landds, Bergen, Norway Abstract. Two types of luciferase that catalyze the luminescent oxidation of coelenterazine were isolated from the marginal exumbrella epithelium (lappet) and the ovary of Periphylla periphylla; they were designated lu-ciferase-L and luciferase-0, respectively. Luciferase-L (Mr 32.000), probably derived from highly specialized photocytes, was very resistant to heat, and its activity was little affected by boiling; but it was unstable in solutions of low ionic strength if bovine serum albumin was not included in the solvent. Luciferase-O (M, 75,000) oc-curred in the eggs in association with particulate matter, and was solubilized and extracted with a buffer containing 2 M guanidine hydrochloride; the enzyme was highly stable in this strongly denaturing solvent. The intensities of the coelenterazine luminescence catalyzed by both lu-ciferases were maximal at pH 7.8 and in the presence of about 1 M NaCl. The quantum yield of coelenterazine was estimated to be 0.14 with luciferase-L (emission max. at 465 nm) and 0.12 with luciferase-0 (emission max. at 470 nm). The luminescence caused by both luciferases was strongly inhibited by Cu"^ and thiol compounds. Introduction All three classes of the phylum Cnidaria contain biolu-minescent species. In the class Hydrozoa, all reported cases of luminescence are caused by Ca-^-sensitive pho-toproteins such as aequorin, found in the jellyfish Ae-quorea aequorea (Shimomura et ai, 1962), and obelin, obtained from the hydroid Obelia sp. (Morin and Has-tings, 1971a, b; Campbell, 1974; Visotskii et ai, 1989). Received 19 September 1997: accepted 17 March 1998. * To whom correspondence should be addressed. E-mail:
[email protected] In the class Anthozoa, light emission is produced by lucif-erin-luciferase type reactions, such as those of the sea pansy Renilla sp. (Cormier, 1978), the sea cactus Cav-ernularia obesa. and the sea pen Ptilosarcus gruneyi (Shi-momura and Johnson, 1979). The bioluminescence of the class Scyphozoa has never been biochemically studied. In the phylum Ctenophora, many species are biolumines-cent, and two of them — Mnemiopsis sp. and Beroe ovata — contain Ca'^-sensitive photoproteins, mnemiop-sin and berovin, respectively (Ward and Seliger, 1974a, b). These proteins are photosensitive and inactivated by exposure to visible light; thus they are distinctly different from the hydrozoan photoproteins. The luminophore of the photoprotein-based biolumi-nescence systems of hydrozoans and ctenophores is coel-enterazine (Anctil and Shimomura. 1984; Shimomura, 1985). Moreover, this same compound serves as the lucif-erin (substrate) in the coelenterazine-luciferase system of the luminescent anthozoans (Shimomura and Johnson, 1975; Cormier, 1978). Although the coelenterazine-lucif-erase system also occurs in many kinds of luminous or-ganisms, including fishes, shrimps, copepods. squids, and coelenterates (Shimomura et ai, 1980: McCapra and Hart, 1980; Campbell and Herring, 1990), coelenterazine luciferase has been isolated and investigated in detail from only two of them; i.e., the sea pansy Renilla (Matthews et ai, 1977) and the decapod shrimp Oplophorus (Shimo-mura et ai, 1978). In a recent study, the luminescence of the scyphozoan jellyfish Periphylla periphylla was found to be associated with two distinct sources: one represented by minute, iiregularly shaped cytoplasmic granules in the cortical layer of maturing ovarian eggs; the other represented by clusters of even smaller, mostly spherical grains within the cytoplasm of highly specialized photocytes that are 244
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