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Comp. Biochem. Physiol., VoL65B,pp. 435 to 437 0305-0491/80/0201-0435502.

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© PergamonPress Ltd 1980.Printedin Great Britain

WIDESPREAD OCCURRENCE OF COELENTERAZINE


IN MARINE BIOLUMINESCENCE
OSAMU SHIMOMURA1, SHOJI INOUE2, FRANK H. JOHNSON1
and YATA HANEDA3
~Department of Biology, Princeton University, Princeton, NJ 08544, U.S.A.,
2Faculty of Pharmacy, Meijo University, Tenpaku, Nagoya 468, Japan and
3Yokosuka City Museum, Yokosuka 238, Japan

(Received 23 April 1979)

Abstraet--l. Coelenterazine is a compound having a key role in the light-emitting process of biolumines-
cent coelenterates. Present evidence concerning the occurrence of coelenterazine and luciferase in other
types of marine organisms establishes that coelenterazine is required for bioluminescence of various,
distantly related, types of organisms such as squids, shrimps and fishes.
2. In the bioluminescent shrimps and fishes, coelenterazine occurs most abundantly along the digestive
tract, suggesting the possibility that this compound derives from ingested food.

INTRODUCTION is required for the bioluminescence of various, dis-


tantly related types of marine organisms, such as
It was generally thought, until a few years ago, that squids, shrimps, fishes and possibly many others.
different types of bioluminescent organisms contain
chemically different luciferins (Glass, 1961). Excep-
tions to this idea, however, were indicated as early as MATERIALS AND METHODS
1961, when evidence was discovered for the chemical All specimens were obtained from Suruga Bay, Japan,
identity of the luciferins of the ostracod crustacean except Watasenia which was from Toyama Bay. The speci-
Cypridina and certain fishes (Johnson et al., 1961 ;Sie mens were stored frozen, mostly at -70°C, for periods of 1
et al., 1961 ; Cormier et al., 1967). These exceptions led month to 1 yr before use. All specimens were dissected
to a new trend of thought regarding the widespread immediately after thawing, and contents of esophagus and
occurrence of luciferins (Cormier et al., 1967; Hori et stomach were removed and discarded. The major organs
al., 1977) which is strongly supported by the present were separately extracted and then tested for the content of
coelenterazine and also for the activity of luciferase. To test
data. luciferase activity, a sample was ground in 0.01 M
Coelenterazine, a compound bearing structural Tris-HCl buffer, pH 8.0, containing 0.1 M NaC1, then cen-
similarity to Cypridina luciferin (Kishi et al., 1966) by trifuged cold. A small portion of the supernatant was
containing an imidazopyrazine skeleton, is the luci- diluted with the same Tris buffer at 25°C making the total
ferin or, sometimes, the key component of a photo- 5 ml, then the luminescence reaction was started by adding
protein in various bioluminescent coelenterates, such 10#1 of a methanolic solution of synthetic coelenterazine
as jellyfish, sea pansies and sea pens (Cormier et al., (Inoue et al., 1975) (85/tg/ml). The intensity of light was
1974; Shimomura & Johnson, 1975). The light emis- measured.
sion of this compound, catalyzed by a luciferase or by To assay the content of coelenterazine, a sample was
ground with approx 10vol of cold methanol and centri-
the protein moiety of a photoprotein, takes place fuged. A small portion of the supernatant (10--100/zl) was
according to the following general scheme:

- ~ N OH

M o ~ N ~ 02
>HO + C02 + Light

Coelenterazine Coelenteramide

Coelenterazine was previously found in the decapod


shrimps Oplophorus and Heterocarpus (Inoue et al., mi;~ed with 5 ml of Tris buffer (the same as above) contain-
1976), the squid Watasenia (Inoue) et al., 1975), and the ing 40/ag of purified Oplophorus luciferase (Shimomura et
fish Neoscopelus (Inoue et aL, 1977). The same com- al., 1978) at 25°C, measuring the total light emitted. Under
the same conditions, 1 ng of pure synthetic coelenterazine
pound was believed to occur also in several other emitted 4.2 x 101~ photons. To detect the possible pres-
species of luminous organisms (Hori et al., 1977). Pre- ence of coelenterazine in a stabilized form, such as coelen-
sent data, concerning the contents of coelenterazine terazine enol-sulfate (Inoue et al., 1977), an identical por-
and luciferase in 16 species of bioluminescent tion of the methanolic supernatant was mixed with an
organisms, conclusively indicates that coelenterazine equal vol of 1 N HCI, heated under argon gas at 90°C for
435
436 OSAMU SHIMOMURAet al.

Table 1. Marine bioluminescent organisms, other than coelenterates, that contain coelenterazine and luciferase

Average . a
body Luciferase actlvity Coelenterazine a'b
Organism
weight (I0 I0 photons/sec) (ng)
(g)

Teuthoidean squid
Chiroteuthis imperator 150 13 (i.o., mantle) 3,000 (liver)
Eucleoteuthis luminosa 80 7 (i.o., mantle) 6,000 (liver)
Onychoteuthis banksi c 55 4,300 (liver) 1,500 (liver)
borealijaponiea
Watasenia 8cintillans i0 0 50,000 (liver)
Sepioidean squid (cuttlefish)
Sepiolina nipponensis 5 1.3 (mantle) 2 (liver)
Decapod shrimp
Sergeste8 lucen8 0.4 0.I (whole body) 4 (hep., stom.)
Sergestes prehensilis i 200 (hep.) 0.03 (stom.)
Systellaspis lanceocaudata 2.1 1,300 (hep., stom.) 700 (stom.)
Oplophoru8 gracilorostris 2.3 5,000 (stom., hep.) 2,000 (stom., hep.)
Heterocccrpus sp. d 14 20,000 (hep., stom.) 5,000 (stom.)
Mysidacean shrimp
Gnathophausia longispina 0.5 70 (hep., stom.) 3/45 (stom.)
Myctophid fish
3,000/7,000
Diaphus elucerm 32 7 (i.o., skin) (p.c., liver)
2,000/5,000
Diaphus coeruleu8 18 6 (skin) (p.c., liver)

Neoscopelus microchir 30 0.3 (skin) 30,000 (liver)


Stomiatoidean fish
Argyropelecus hemigymnus 3 0.2 (skin) 20 (stom.)
Yarrella illustris 25 2 (skin) 0.5/10 (p.c.)
i

a Sum for one whole animal excluding the contents of stomach and esophagus. Main locations of luciferase
and coelenterazine in the body are shown in parentheses, with abbreviations as follows: 1.o., light organ; hep.,
hepatopancreas; stom., stomach; p.c., pyloric caeca. Stomachs of shrimps were tested together with reddish
tissue surrounding the stomachs.
b The assay value after the acid treatment is given after a virgule only when it was different from the one
before such treatment, and the difference of the two values represents the'amount of a stabilized derivative of
coelenterazine, probably the enol-sulfate.
c Data on a single specimen. The other data was based on the results from more than five specimens.
d Species unconfirmed. It was seemingly different from the organism that was previously referred to as
H. laeviqatus by Inoue et al. (1976).

l min, cooled, neutralized with NaHCOa, and finally 80-90Vo of b o t h coelenterazine and luciferase activi-
assayed for the total amount of coelenterazine as described ties were found in the thorax, most abundantly in the
above. hepatopancreas, as well as a r o u n d the stomach and
near the base of the legs. Several unidentified species
RESULTS AND DISCUSSION
of bioluminescent decapod shrimp and myctophid
The results are summarized in Table 1. With the fish, not listed in the table, yielded results similar to
exception of Watasenia, some luciferase activity was the data included in Table 1, but several kinds of
found in practically every part of the body of each non-bioluminescent squids, shrimps and fishes yielded
species, not only in the locations listed in Table 1. As negative results throughout.
for coelenterazine, some, at least a trace, was always All the assays shown in Table 1 should be con-
found in the light organs. A large a m o u n t of the same sidered m i n i m u m values, because spontaneous de-
compound, however, was often found also in other creases in amounts of both coelenterazine and lucifer-
organs, e.g. the liver of squids and the liver and ase activities take place during storage, even in a
pyloric caeca of myctophid fishes. In the shrimps, frozen state. Variation of activities due to seasonal or
Coelenterazine in bioluminescence 437

other causes may also occur. In fact, one of us (Y.H.) (crustacean). Biochem. biophys. Res. Commun. 29,
once observed brightly luminescent clouds ejected 747-752.
into sea water from living specimens of Sepiolina. The CORMIER M. J., HORI K. & ANDERSONJ. M. (1974) Biolu-
present Sepiolina specimens, however, were only minescence in coelenterates. Biochem. biophys. Acta 346,
137-164.
dimly luminescent when alive, and none were
GLASSB. (1961) In Light and Life (Edited by McELROV W.
observed to eject any luminous cloud, D. & GLASSB.), pp. 849-850. Johns Hopkins Press, Bal-
The structural identification of coelenterazine in timore.
Watasenia, Oplophorus, Heterocarpus and Neoscopelus HANEDA Y., JOHNSONF. H. & SHIMOMURAO. (1966) The
has been previously reported (Inoue et al., 1975, 1976, origin of luciferin in the luminous ducts of Parapriacan-
1977). By similar methods we have now established the thus ransonneti, Pempheris klunzingeri, and Apooon
presence of the same compound in Chiroteuthis, Euc- ellioti. In Bioluminescence in Progress (Edited by JOHN-
leoteuthis, Diaphus elucens and Diaphus coeruleus. For SON F. H. & HANEDAY.), pp. 533-545. Princeton Univer-
the rest of the species listed in Table 1, the structure is sity Press, Princeton, N.J.
HORI K., CHARBONNEAUH., HART R. C. & CORMIERM. J.
very likely identical; if any modification of the struc-
(1977) Structure of native Renilla reniformis luciferin.
ture occurs, it must be very minor, considering the Proc. hath. Acad. Sci. U.S.A. 74, 4285-4287.
specificity of Oplophorus luciferase used in the assay of INOUE S., SUGIRAS., KAKOI H., HAS1ZUMEK., GOTO T. &
this compound. Thus the bioluminescence of every hO H. (1975) Squid Bioluminescence II. Isolation
species listed in Table 1 must involve either coelenter- from Watasenia scintillans and synthesis of
azine itself or a slightly modified form of its usual 2-(p-hydroxybenzyl)-6-(p-hydroxyphe n yl)- 3,7-dih ydr oi-
structure. Moreover, in all instances except Watasenia midazo(1,2-a)pyrazin-3-one. Chem. Lett. 141-144.
in which luciferase activity was not found, the funda- INOUES., KAKOIH. & GOTO T. (1976) Oplophorus luciferin,
mental role of this compound is that of a luciferin. In bioluminescent substance of the decapod shrimps, Oplo-
Watasenia, coelenterazine has been reported to be a phorus spinosus and Heterocarpus laevigatus. Chem. Com-
mun. 1056-1057.
precursor of the functional luciferin (Inoue et al.,
INOUE S., KAKOI H. & GOTO T. (1976) Squid biolumines-
1976). cence III. Isolation and structure of Watasenia luciferin.
The rich occurrence of coelenterazine along the Tet. Lett. 2971-2974.
digestive tract of the shrimps and fishes suggests the INOUE S., KAKOI H., MURATAM., GOTO T. & SHIMOMURA
possibility that this compound derives from ingested O. (1977) Complete structure of Renilla luciferin and
food, in some such manner as previously proposed for luciferyl sulfate. Tet. Lett. 2685-2688.
Cypridina luciferin found in the fishes Parapriacanthus INOUE S., OKADA K., KAKOI H. & GOTO T. (1977) Fish
and Porichthys (Haneda et al., 1966; Tsuhi et al., bioluminescence I. Isolation of a luminescent substance
1972); in fact, the esophagi of the present fish speci- from a myctophina fish, Neoscopelus microchir, and iden-
tification of it as Oplophorus luciferin. Chem. Lett.
mens were often filled with small shrimps, including
257-258.
Sergestes lucens, as well as unidentified other species. JOHNSON F. H., SUGIYAMAN., SHIMOMURAO., SAIGAY. &
If coelenterazine is obtained only from food, then it HANEDA Y. (1961) Crystalline luciferin from a lumi-
would be very interesting to know the ultimate source nescent fish, Parapriacanthus beryciformes. Proc. hath.
in the food chain wherein this compound is synthe- Acad. Sci. U.S.A. 47, 486-489.
sized. KISHI Y., GOTO T., HIRATAY., SHIMOMURAO. • JOHNSON
Considering the variety of types of organisms which F. H. (1966~I Cypridina bioluminescence I. Structure of
contain coelenterazine, it seems very probable that an Cypridina luciferin. Tet. Lett. 342%3436.
even more general importance is to be attributed to a SmMOMURAO. & JOHNSONF. H. (1975) Chemical nature of
bioluminescence systems in coelenterates. Proc. Natn.
fundamental role of coelenterazine in the light-emit-
Acad. Sci. U.S.A. 72, 1546-1549.
ting systems of a major part of all known biolumines- SHIMOMURAO., MASUGIT., JOHNSONF. H. 8~ HANEDAY.
cent organisms. (1978) Properties and reaction mechanism of the biolu-
Acknowledgements--We thank Dr M. Omori for identi- minescence system of the deep-sea shrimp Oplophorus
f~cing some of the organisms. This work was supported by gracilorostris. Biochemistry 17, 994-998.
NSF Grants PCM76-12301 and 01P75-15386 and NIH SIE E. H. C., MCELRoY W. D., JOHNSONF. H. & HANEDA
Grant GM25093. Y. (1961) Spectroscopy of the Apogon luminescent sys-
tem and of its cross reaction with the Cypridina system.
REFERENCES
Archs Biochem. Biophys. 93, 286-291.
TSUJI F. I., BARNESA. T. & CASEJ. F. (1972) Biolumines-
CORMIER M. J., CRANEJ. M. JR & NAKONOY. (1967) Evi- cence in the marine teleost, Porichthys notatus, and in-
dence for the identity of the luminescent systems of Por- duction in a non-luminous form by Cypridina (ostracod)
ichthys porosissimus (fish) and Cypridina hilgendorfii luciferin. Nature 237, 515-516.

c.a.p. 65/2n~O

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