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Chemical Nature of Bioluminescence Systems in Coelenterates (Photoprotein/Luciferin/Luciferase/Aequorea/Renilla)

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Chemical Nature of Bioluminescence Systems in Coelenterates (Photoprotein/Luciferin/Luciferase/Aequorea/Renilla) Proc. Nat. Acad. Sci. USA Vol. 72, No. 4, pp. 1546-1549, April 1975 Chemical Nature of Bioluminescence Systems in Coelenterates (photoprotein/luciferin/luciferase/Aequorea/Renilla) OSAMU SHIMOMURA AND FRANK H. JOHNSON Department of Biology, Princeton University, Princeton, New Jersey 08540 Communicated by Arthur B. Pardee, February 10, 1976 ABSTRACT Analysis of substances involved in light- OH emitting reactions among bioluminescent coelenterates Ca2' has revealed a pronounced uniformity in the structural -a OOH features of initial reactants, i.e., "luciferins" and photo- IProteinh protein chromophores, as well as the light-emitter prod- Wi---YCI uct. This product is structurally identical among the dif- ferent classes of coelenterates: Hydrozoa (the jellyfish, -CHO Aequorea), Anthozoa (the sea cactus, Cavernularia; sea Protein + Ia + CO2 + Light pansy, Renilla; and sea pen, Leioptilus), and very likely -H2 also the Scyphozoa (the jellyfish, Pelagia). In each of these -YC instances the reaction product, namely, 2-(p-hydroxy- phenylacetyl)amino-3-benzyl-5-(p-hydroxyphenyl) pyra- In the bioluminescence of the anthozoan Renilla (sea pansy) zine, is the actual light-emitter, whether it occurs in a Ca2 t-triggered photoprotein type of luminescence, or in a an unstable luciferin (IIIb), having an unknown group (X), "luciferin-luciferase" type. The evidence indicates that in reacts with molecular oxygen in the presence of Renilla luci- certain coelenterates, e.g., Cavernularia, these two types ferase, producing light, C02, and compound Ib (6, 7). Renilla are equally significant, whereas in others (Renilla and luciferin (IIIb) is stored in the organism in the form of a de- Leioptilus) the "luciferin-luciferase" type predominates "luciferyl sulfate" (IIb), which is more over the Ca-triggerable photoprotein type, and finally that rivative, termed only the photoprotein type functions in the luciferaseless stable than IIIb, but which can be converted to IIIb either by jellyfish, Aequorea. In all instances investigated, the struc- 3',5'-diphosphoadenosine in the presence of luciferin sulfo- ture of the light-emitter prior to the luminescence reac- kinase, or by treatment with acids (8-10). tion appears to be essentially the same as that of the Many of the luminescent coelenterates have been reported chromophore of unreacted aequorin. The product of the sul- luminescence reaction is absent in extracts of nonlumi- to contain one or more of the following factors: luciferyl nous species. However, a product very similar to that of fate which is closely similar in chemical nature to that found in luminescent coelenterates occurs also in representatives of Renilla, luciferase, luciferin sulfokinase, and a photoprotein other phyla, including the cephalopod molluses, e.g., the (11, 12). Since the anthozoans Renilla, Cavernularia (sea cac- "firefly squid" Watasenia and probably various cteno- contained all of these factors, as well. tus), and Leioptilus (sea pen) phores the involvement of both types of luminescence mechanisms In the bioluminescence of the hydrozoan jellyfish, Aequorea are implied, i.e., photoprotein and luciferin-luciferase types in aequorea, the photoprotein aequorin reacts with Ca2+, either these species. In addition to the photoprotein aequorin, the in the presence or absence of molecular oxygen, resulting in the jellyfish, Aequorea, was found to contain luciferyl sulfate but emission of -blue light by an intramolecular reaction (1, 2). no luciferase (11). In Cavernularia, the light-emitting molecule The light-emitting molecule involved has been identified as Ia has been identified to be Ia (13), the same as in Aequorea. (Fig. 1 and ref. 3). In Aequorea, a further process of an inter- Recently, a new protein in Renilla has been reported (14). molecular energy transfer takes place in vivo between the This protein, which when bound with luciferin releases the excited state of Ia and a green fluorescent protein, the latter bound luciferin on addition of Ca2+, was termed "luciferin ultimately emitting a green light (4). The present study, how- binding protein," and the existence of analogous proteins ever, is limited to the initial light-emitting reaction of Aequo- among other luminescent anthozoans has been presumed rea, as well as all other luminescent species of coelenterates (14). Thus, the plotoprotein activity in Renilla (11, 12) could that have been investigated from this point of view. be accounted for, at least to some extent, by the presence of In the aequorin reaction, the structure of the light-emitting the luciferin binding protein. However, the existence of any molecule (Ia) prior to the luminescence reaction has been photoproteins in Renilla and other anthozoans still remains to elucidated (Ha, i.e., the enolized form of lIla bound to a pro- be proved. tein), and the intramolecular reaction involved in this lumi- The present study was undertaken in an effort to identify nescence was postulated to occur as shown in the following the structures of light-producing compounds, such as luci- overall scheme (5) wherein an essential component, H202, is ferin, and the light-producing chromophore of a native photo- tentatively assumed to be in the form of an a-hydroxyhydro- protein, as well as the structures of light-emitters derived a chromophore which has therefrom, in representative coelenterates. Comparisons have peroxide and YC refers to yellow of been isolated but has a yet unknown structure. also become possible regarding the quantitative extents 1546 Proc. Nat. Acad. Sci. USA 72 (1M) Bioluminescence Systems in Coelenterates 1547 photoprotein activity, as well as content of luciferyl sulfate R and light-emitting compounds produced by in vivo lumines- Ia R =-C-CM2 OH cence. N NH llb R= -C-X MATERIALS AND METHODS HO) /e HI Aequorea aequorea, Leioptilus, and a nonluminescent pen- Ic R= -H natulid resembling Stylatula were obtained at or from the Friday Harbor Laboratories of the University of Washington, and Cavernularia obesa at the Uozu Aquarium and Amakusa Marine Laboratory, Japan. Renilla malleri and Leptogorgia lha RI = -CH2 JOH virgulate were purchased from Gulf Specimen Co., Panacea, R2 =-protein Fla., and Renilla kollikeri from the Pacific Bio-Marine Supply Ab = -X Co., Venice, Calif. The animals were generally kept in aerated RI R2= -SOj (artificial) sea water at 13-15° until ready for use. Before extraction and measurement of various factors, ]IC R, C 2~ O Cavernularia (whole body), Leioptilus (after removing the R2= -50 nonluminescent stalk and horny axis), and Renilla (after re- moving stalk), all in contracted states, were cut to slices 3 mm thick with a sharp razor blade. Stylatula-like species (also after removing the horny axis) and Leptogorgia were chopped into pieces 1 cm in length. With Aequorea, thin strips (2-3 mm Na R= -CH2Q OH thick) which contained luminescent organs were cut off from the margin of the umbrella, and only these strips were used. INfb R= -X Most extractions were carried out at night, between 8 and 12 p.m., due to insufficient knowledge concerning the day-night FIG. 1. Structures of compounds involved in luminescence of rhythm of some of these bioluminescent systems. coelenterates, as discussed in the text. Measurement of In Vivo Luminescence. To a test tube con- taining 1 ml of sea water and a small piece of test sample showing a blue fluorescence, was finally evaporated to dry- taken from various parts of the specimen jody, 2 ml of 1 M ness. The dried residue was extracted with less than 1 ml of KCl was added at 200, and the resulting luminescence was methanol, the insoluble matter discarded, and the methanol- measured until the light finally ceased, usually within 10 min. soluble material was further purified by two steps of thin- The emitted light was measured by means of a photomulti- layer chromatography on silica gel, first with water-saturated plier-light integrating amplifier-recorder assembly which was ether (RF of Ia; 0.5) and then with water-saturated chloro- calibrated in absolute units at various wavelengths. form ethylacetate (1:1) (RF of Ia, 0.36). Measurement of Ca2+-Triggered Luminescence Activity. The amount of compound Ia obtained by the above pro- Slices or pieces of the specimen (5 g) were added to 20 ml of cedure was estimated from the absorption spectrum mea- 0.05 M EDTA (pH 9.0 at 200, adjusted with Tris) at 00. After sured in methanol, taking an e value of 13,500 for the 333 rum leaving 10 min, the pieces in the solution were repeatedly absorption peak. pressed and squeezed with a spatula to discharge most of the Extraction of Luciferyl Sulfate (fIb), Estimation of the potential light-emitting activity into the solution. This ac- Amount of Luciferyl Sulfate by Chemiluminescence after Acid- tivity was then measured by adding 2.5 ml of 0.02 M calcium Treatment of the Sample, and Isolation of the Product of the acetate to 0.5 ml of the test solution at 200. Chemiluminescence Reaction. To avoid luminescence of living Isolation of Compound Ia after Stimulating Specimens to specimen, specimens in sea water, except for Aequorea, were Luminescence In Vivo. The method described here is in prin- cooled in advance slowly to near 0°, before the specimens were ciple the same as that which was previously used for Cavernu- cut into slices. Strips containing the luminescent organs of laria (13), although a few improvements in purification pro- Aequorea, or slices of other species prepared after cooling cedures were made because of greater amounts of impurities (about 80 g), were added into 300 ml of methanol at 00. After encountered in Leioptilus and Renilla. leaving 15 min, the pieces in methanol were pressed and Slices or pieces of specimen (80 g) were put into 80 ml of 1 M squeezed using a spatula and a pair of pliers, then these pieces KCl containing 0.01 M CaCl2 to stimulate in vivo lumines- were broken down to smaller pieces by brief use of a Servall cence.
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