T h e r e h a v e b e e n r e c e n t a n d s i g n i f i c a n t a d v a n c e s in o u r u n d e r s t a n d i n g o f t h e
m o l e c u l a r ba s i s f o r b i o l u m i n e s c e n c e a n d i ts c o n t r o l in m a r i n e c o e l e n t e r a t e s . I t is t h e
p u r p o s e o f th i s r ev i ew to o u t l i n e t he se a d v a n c e s w h i c h h a v e o c c u r r e d o v e r t h e p a s t
twe lve yea r s in seve ra l l a b o r a t o r i e s .
T h e b i o l u m i n e s c e n t o r g a n i s m s s t u d i e d o c c u r in t he p h y l a Cnidaria a n d Cteno-
phora a n d a c o m p l e t e l i s t ing o f t h e species i n v e s t i g a t e d c a n be f o u n d in T a b l e 1.
T h i s t a b l e a l so l ists c e r t a i n c h a r a c t e r i s t i c s o f t he l u m i n e s c e n t s y s t e m o f e a c h o r g a n i s m
TA
BL
E
I
A
LIS
T
OF
B
IOL
UM
INE
SC
EN
T
CO
EL
EN
TE
RA
TE
S
INV
ES
TIG
AT
ED
A
ND
G
EN
ER
AL
C
HA
RA
CT
ER
IST
ICS
O
F
TH
EIR
L
UM
INE
SC
EN
T
SY
ST
EM
S
Th
e b
lan
k s
pac
es i
nd
icat
e th
at t
he
char
acte
rist
ic f
eatu
re i
n q
ues
tio
n h
as n
ot
bee
n e
xam
ined
.
An
imal
s ex
amin
ed
Pre
sen
ce o
f R
enil
la-l
ike
Lu
cife
ryl
Lu
cife
rin
- S
ulf
o-
sulf
ate
bin
din
g
kin
ase
pro
tein
ac
tiv
ity
I.
Ph
ylu
m C
nid
aria
A.
Cla
ss A
nth
ozo
a:
Ren
illa
reni
form
is
Ren
illa
mM
leri
R
enill
a ko
klik
eri
Cav
ernu
lari
a ob
esa
Piil
osar
cus
guer
neyi
St
ylat
ula
e[on
gata
A
cant
hopt
ilum
gra
cile
--
P
araz
oant
hus
luci
ficum
B.
Cla
ss H
yd
rozo
a:
Aeq
uore
a ae
quor
ea
Aeq
uore
a fo
rska
lea
Hal
ista
ura
sp.
Obe
lia l
ongi
ssim
a O
belia
gen
icul
ata
Obe
lia b
icus
pida
ta
Obe
lia c
omrn
isur
alis
C
lyti
a ed
war
dsia
C
ampa
nula
ria
calc
oelif
i'ra
Phi
alid
ium
sp.
C.
Cla
ss S
cyp
ho
zoa:
P
elag
ia n
octil
uca
Ox
yg
en
Em
issi
on
Lu
cife
rase
G
reen
- -
Lum
i~so
mes
re
qu
ire-
m
axim
a (n
mt
acti
vit
y
flu
ore
scen
t m
ent
in v
ivo
in
vit
ro
pro
tein
(i
n v
itro
)
2
~ 5
09
4
90
4
509
49
0
. 4
: 50
9 4
90
-
509
49
0
_ 4
~ 50
9 4
90
_
4
~ 50
9 4
90
•
4 ~
509
49
0
_1
7
"2
m
"2
T-
--]
46
9 ~
'2
50
82
46
9
1
4 e
5082
47
52
.4
'2
508
~ 47
52
._-2
50
82
• 2 50
82
_ 4
2 50
82
o
2 50
8 ~
2 "2
II.
Phy
lum
Cte
no
ph
ora
A.
Cla
ss T
enta
cula
ta:
Mne
rnio
psis
lei
dyi
Mne
mio
psis
sp.
+
B.
Cla
ss N
ud
a:
Ber
oY o
vata
1 S
him
om
ura
et
al.
(ref
s 2,
63)
. 2
Mo
rin
and
Has
ting
s (r
ef.
42).
3
War
d a
nd
Sel
iger
(re
f. 8
4).
2 e
4852
48
52
__3
3 48
83
4853
:3
3 49
43
4853
4 Id
enti
cal
char
acte
rist
ics
to R
enill
a re
nifo
rmis
lu
mis
omes
wit
h th
e fo
llow
ing
exce
pti
on
s: T
hese
wer
e n
ot
exam
ined
by
elec
tron
mic
rosc
op
y a
nd
th
e H
yd
rozo
an l
umis
omes
did
no
t re
quir
e ox
ygen
fo
r lu
min
esce
nce.
In
add
itio
n,
the
Hy
dro
zoan
lum
isom
es d
o n
ot
con
tain
a l
ucif
erin
bin
ding
pro
tein
li
ke t
hat
fou
nd
in
the
An
tho
zoan
lum
isom
es.
140
to be referred to in subsequent discussions. Certain of the bioluminescent systems listed in Table I, such as that of Renilla, are now reasonably well understood at the molecular level. It has also become increasingly clear that the chemical path to light emission is similar, if not identical, in all marine coelenterates. There are subtle differences but these appear to be related to the manner in which the bioluminescent flash is controlled rather than to basic differences in the chemistry. Based on data published over the past two years it is hoped that this point will be made clear in this review.
In general bioluminescence can be defined as the emission of visible radiation by living organisms or by substances derived from them. Due to the work of Dubois [1 ] the terms luciferin and luciferase have become part of the terminology in the field of bioluminescence. In the classic sense luciferase refers to an enzyme that catalyzes the oxidation of a substrate, luciferin, with light emission. Luciferin can be defined as a reduced compound which when oxidized in the appropriate environment will produce a fluorescent product in the electronic excited state. Chemical energy released during the oxidative reaction is utilized to create an electronic excited state either directly or by energy transfer to some fluorescent species formed, or present, duling the reaction. This fluorescent species, or emitter may be a product of luciferin oxidation, or some protein-bound chromophore in close association with luciferase, depending upon the organism.
In the case of certain coelenterates the classic definitions of luciferase and luciferin would appear not to apply. Thus Shimomura et al. [2] demonstrated that light emission in certain hydrozoans occurred when calcium interacted with a protein referred to as a photoprotein. These photoproteins react only once, i.e. they do not turnover, and have been consistently viewed as unique by Johnson and Shimomura [3] in contrast to the luciferin-luciferase system of other coelenterates such as Renilla [4]. However, evidence accumulated in this laboratory over the past two years has demonstrated that photoproteins are not unique but possess striking chemical simi- larities to the light producing system of Renilla and other coelenterates. The evidence for this is overwhelming, and will represent one of the major points of emphasis in this review.
Work done in several laboratories over the past decade has contributed signifi- cantly to the understanding of the molecular basis for bioluminescence in coelen- terates. Numerous other bioluminescent systems have also been investigated during this period but will not be reviewed here. For additional information the reader is referred to a number of more recent reviews [3-11,75,94].
II. BIOLUMINESCENCE IN THE ANTHOZOANS
IIA. General comments Members of this class represent some of the most beautiful marine creatures in
existence and display a brilliant and striking luminescence. Examples of those whose
141
Fig. 1. The sea pansy, Renilla reniformis. A colonial soft coral (Anthozoan), consisting of rather flat and circular features (about 4 cm in diameter), normally found resting on the ocean bottom.
b ioluminescence has been s tudied at the biochemical level are listed in Table I and
include the sea pansy (Renilla), sea pens (Stylatula and Acanthoptilum), the sea feather
(Ptilosarcus) and Parazoanthus. These animals , consis t ing o f colonies of individual
polyps, p roduce bioluminescence that is cont ro l led by a nerve ne twork [12] inter-
connect ing each polyp. Tacti le or electrical s t imula t ion results in concentr ic waves
of greenish luminescence that travel across the surface and up the polyps o f the animal
at a rate o f abou t 8 cm • s -1 at 21 °C. A pho to g ra ph of the sea pansy (Renilla) is
shown in Fig. 1.
Of all the coelenterates , the molecular basis for b ioluminescence and cont ro l
of the b io luminescent f lash is best unders tood in Renilla. The ReniLla system will
therefore be descr ibed first. The advan tage here is that all o ther coelenterate biolumi-
nescent systems can be more easily explained due to the existing similarit ies to that of
Renilla in the chemical pa th to light emission.
HB. Renilla (Sea Pansy) bioluminescence IIB-I. Chemical requirements for blue light emission. From approx ima te ly
40000 animals , ob ta ined by dredging the ocean bo t tom at depths of 10-20 m, one
142
I I\
O ~ R (tl) Nv N
Renillo LudJerin
f , . , O ~ C H 2 0
!J ")
I-udferin I~ynthetic} ~
H 0
(/ /
Lucileryl Sulfol~
HaQsPO OH PAP
Fig. 2. Structures of native and synthetic substrates involved in Renilla (sea pansy) bioluminescence.
can obtain about 0.5 mg of pure Renilla luciferin. For this reason the elucidation of the structure of this luciferin presented a difficult challenge. A breakthrough in the solution to this problem occurred when a partially active analogue of Renilla luciferin was synthesized by Hori and Cormier [13]. This compound (1 in Fig. 2) was found to be 10 % as active as native luciferin (ll in Fig. 2) in the bioluminescence assay using Renilla luciferase.
From mass spectral data on native luciferin it was deduced that replacement of the methyl group of Compound I by benzyl would result in a compound more closely related to that of native luciferin. This compound (lIl in Fig. 2) was synthesized by Hori et al. [14] and found to be fully active in producing light with luciferase when compared with native luciferin (II). Furthermore, the bioluminescence quantum yield of Compound lII was found to be about the same as that of native luciferin, i.e. approximately 5 ~ . Using Renilla luciferase the wavelength distributions of light emission, as illustrated in Fig. 3, are the same in the presence of either synthetic luciferin or native luciferin. The luminescence is characteristically blue having a broad emission with a maximum at 490 nm. Because either native or synthetic luciferins (ll and III in Fig. 2) behave similarly in their reaction with luciferase the term luciferin will be used throughout to refer to either of these compounds.
Renilla stores luciferin, an easily autooxidizable compound, as a stable sul- fonated derivative termed luciferyl sulfate [15]. The structure of luciferyl sulfate (IV in Fig. 2) has been confirmed by chemical synthesis from luciferin and sulfamic acid [16].
Luciferyl sulfate is not a substrate for luciferase and therefore it must be converted to luciferin. In Renilla this is accomplished by the enzyme luciferin sulfo- kinase (Y-phosphoadenylyl sulfate: luciferin sulfotransferase) in the presence of 3' ,5'-diphosphoadenosine (PAP in Fig. 2) as demonstrated by Cormier and associates [15,17]. The reaction is illustrated below:
Fig. 3. A comparison of the color of bioluminescence upon initiation of the luciferase-catalyzed reaction with synthetic Renilla luciferin (A) and native luciferin (B).
luciferin sulfokinase
Luciferyl sulfate 3 PAP 7----- "i luciferin + PAPS
This reaction is easily reversed since luciferyl sulfate can be produced from luciferin and 3'-phosphoadenosine-5'-phosphosulfate (PAPS).
Luciferase has been purified to homogeneity and has a molecular weight of approximately 24000 [14]. In the presence of oxygen, luciferase will catalyze the oxidation of luciferin resulting in the production of blue light with an emission maximum at 490 nm as shown in Fig. 3. During this luminescent reaction oxy- luciferin and CO2 are the major products formed [14,18] as illustrated below:
luciferase
Luciferin + O2 - - 4 oxyluciferin + CO2 + light
Oxyluciferin was isolated from the reaction mixture and its structure (I in Fig. 4) confirmed by chemical synthesis [14]. In this aqueous environment a small amount of luciferin is autooxidized ( < 1 0 ~ ) via a nonluminous path to yield Renilla etio- luciferin (II in Fig. 4).
In water or in methanol luciferin exhibits a characteristic visible absorption at 435 nmwith a emM 43s"m of 9.7 in methanol [14]. Observations made after biolumines- cence shows that this absorption band disappears with the appearance of a new one at 335 nm characteristic ofoxyluciferin. This is illustrated in Fig. 5. The ~mM T M for oxyluciferin in dimethylformamide was found to be 13.6 [14].
The nature of the electronic excited state responsible for light emission during the oxidation of luciferin was arrived at by studying the chemiluminescence of
144
( I ) ' " (1 ' ~ t, r , i , "
HO l~enilla Oxyludferin ,~;"]i H(
Uxyluflleilfl MonoQnnon ,Syntheli(I
(rO ~N~NFI2
llenillD Eliol~dterin V
( v '
H O Aequereo Oxylouternn
Oxylucite[in Llianlon
Fig. 4. Structures of the major products and emitters of coelenterate in vitro luminescence.
luciferin. Hori et al. [19] reported that when luciferin is dissolved in aprotic solvents such as dimethylformamide a bluish luminescence occurs (2ma, : 480 rim). Oxygen was required for this luminescence and approximately one mole of oxyluciferin (I in Fig. 4) was produced per mole of luciferin utilized [14]. When strong base was added to the dimethylformamide the resulting luminescence was yellow-green (Zm~x =: 538 rim). A study of the spectroscopy of this luminescent reaction using luciferin and a number of its derivatives [14] revealed that the yellow-green emitter was a dianion of oxyluciferin (Ill in Fig. 4) and that the blue emission was due to the electronic excited state of the monoanion of oxyluciferin (IV in Fig. 4). The evidence suggest that the monoanion is formed directly and that its fluorescence decay rate is faster than the rate of protonation in dimethylformamide [14]. These results are consistent with findings made by organic chemists using model compounds (20-22).
WAVELENGTH IN NM
5~ 4~ 3~
"04I C
A .-.
• / 0 20,00O 30,000 40,000
WAVENUMBER IN CM 1
Fig. 5. Absorption spectrum of the Renilla (sea pansy) biolurninescence product (B). of synthetic oxyluciferin (C) and of synthetic luciferin (A).
Fig. 6. Pathway to light emission during both the chemiluminescent and bioluminescent oxidation of synthetic Renilla luciferin.
The evidence suggests [14] that in bioluminescence, as well as in chemilumi- nescence, the electronic excited state responsible for the emission is the monoanion of oxyluciferin (IV in Fig. 4). Thus the pathway to light emission in both bioluminescence and chemiluminescence is viewed as illustrated in Fig. 6. Oxyluciferin and its mono- anion do not fluoresce in an aqueous environment although in dimethylformamide they are highly fluorescent with quantum yields of 23 and 6 ~ , respectively [14]. Since the bioluminescence quantum yield is 5 ~, and no hint of a contribution by the neutral species of oxyluciferin occurs during bioluminescence, it appears that light emission is derived from the excited state of the luciferase-oxyluciferin monoanion complex. Apparently dimethylformamide and luciferase provide similar environments resulting in a fluorescence quantum yield of 5 - 6 ~ for the emitting species in each case. The chemiluminescence quantum yield of luciferin in dimethylformamide, however, is only 0.1 )/o. Thus it appears that one of the interesting features of Renilla
luciferase is its ability to generate electronic excited states in yields approaching 100 ~. Firefly luciferase also exhibits this interesting feature [23].
IIB-2. Mechanism studies on the light reaction. The mechanism of the luciferase catalyzed bioluminescent oxidation of luciferin has been studied with l SO-labeled water and oxygen [24]. The COz labeling patterns were consistent with the postulated scheme shown in Fig. 7 (Pathway A) since oxygen in the COz produced was derived from water, not 02. These results are also consistent with analogous studies done on the firefly bioluminescent reaction [25].
However, similar studies on the mechanism of Cypridina bioluminescence are accounted for by Pathway B (Fig. 7) since one of the oxygens of CO2 is apparently derived from molecular oxygen, not water [26]. Shimomura and Johnson [27]
146
')i brr<, N . . r x
~[' rx~R.: tuclreR~N
- Iq
m~ t~ 3 B
O, 0 ~
NGN: :~ j -N~ ,,,:v
N N ® /OXYLUCIFERIN J f T JMo,o,N,o,
1 OXYLUCIFERIN + LIGHt
Fig. 7. Proposed oxidative mechanisms during the bioluminescent oxidation of Renilla luciferin
attempt to explain the conflicting data by showing that the ~SO-labeling pattern is dependent upon Cypridina luciferin concentration during bioluminescence. The data suggest that one oxygen from 02 is incorporated into CO2 at high, but not low, luciferin concentrations suggesting that oxygen exchange between water and CO2 occurs at low CO2 concentration. In contrast, studies on the chemiluminescence of firefly luciferyl adenylate [28] have demonstrated that the ~80-labeling patterns are in accord with those observed during bioluminescence, thus the oxidative path was postulated to proceed by a route analogous to Pathway A (Fig. 7). The same results have been obtained at low or high luciferyl adenylate concentrations suggesting that CO2 concentration is not critical in this case (DeLuca, M. and Dempsey, M., personal communication).
For a clear understanding of the oxidative route to bioluminescence or chem- iluminescence an important criterion must be met; this entails an adequate explanation of the 1 SO.labeling patterns coupled with a mechanism which provides an appropriate theoretical explanation for the generation of excited states. The postulated linear hydroperoxide, derived from oxygen attack on a carbanion, represents a key inter- mediate in the scheme shown in Fig. 7. An analogous intermediate was originally postulated by several investigators working with model compounds who suggested Pathway B (Fig. 7) as the route to chemiluminescence [20,29-32]. This route is presumed to proceed via the formation of a cyclic oxygen-containing intermediate (dioxetanone) analogous to the one shown in Fig. 7 although this pathway has not been confirmed by ~80 studies with the model compounds. This proposal is at- tractive, however, since it provides a theoretical basis for the formation of electronic excited states [33]. Using model systems, similar cyclic intermediates have been synthesized [34-39] but their decompositions yield triplet, not singlet, excited states.
147
MBvel#n~/b (NM) $oo ~oo
Ai~ OO0 ~0 0OO
Fig. 8. Comparison between the in vivo bioluminescence emission of Renilla (upper) and the fluo- rescence emission of the green fluorescent protein (lower).
However, in bioluminescence we are dealing with excited states of singlet multiplicity. It is clear that an understanding of the oxidative mechanisms involved in
bioluminescence and chemiluminescence requires further work. It also appears that water is involved in the oxidative path to light emission in Renilla and the firefly. Thus the results with Cypridina luciferin are surprising since the structures of Renilla and Cypridina luciferins are similar [13]. One would therefore expect their chemical behavior to be the same although it is not inconceivable that two different pathways could give rise to an excited state product.
HB-3. Requirements for green light emission. Qualitative differences in the color of light produced in vitro and in vivo by Renilla and a number of other coelen- terates were reported by Hastings and Morin [40] and by Morin and Hastings [41,42]. They reported that the in vitro reactions were distinctly bluish whereas the in vivo emissions were green. A more precise comparison was made by Wampler et al. [43] who showed that although Renilla luciferase catalyzes the oxidation of luciferin with subsequent emission of blue light as shown in Fig. 3 the live animal produces green light characterized by a very narrow structured emission (2ma X -: 509 rim) as shown by the upper curve in Fig. 8. The difference between the in vitro and in vivo emissions was presumed to be due to a green fluorescence noted in the tissues of Renilla and
148
certain other coelenterates by Morin and Hastings [42]. In addition, Morin and Reynolds [95,96] have demonstrated an exact correspondence between the luminescent and green fluorescent sites in Renilla and in several Hydrozoans.
This green fluorescence in Renilla was found by Wampler et al. [43] to be due to a green fluorescent protein which they originally observed to be carried along during the purification of luciferase. They subsequently purified this protein to homogeneity and have studied a number of its characteristics [43,44]. This green fluorescent protein contains a chromophore of unknown structure which is apparently covalently linked to the protein. Its fluorescence quantum yield was found to be 30 ~ [43 ].
The significance of this green fluorescent protein is indicated by comparison of its fluorescence emission with the in vivo bioluminescence of Renilla as illustrated in Fig. 8. Like bioluminescence the fluorescence of the green fluorescent protein exhibits a narrow, structured emission with a maximum at 509 nm [43]. A detailed spectral analysis has suggested that the absorption and emission transitions of this protein arise from the same electronic transition and that the structure seen at 540 nm is of a vibrational nature and not due to electronic or species differences [43]. The near identity of the emissions in Fig. 8 is evident. The only minor difference occurs in the blue region of the spectrum where bioluminescence, but not fluorescence, makes a contribution. When the difference spectra in this blue region was determined it was found to be identical to the luciferase-catalyzed blue emission shown in Fig. 3 [43]. Thus during in vivo bioluminescence in Renilla there are two emitters. One is derived from the electronic excited state of the oxyluciferin monoanion (IV in Fig. 4) and its contribution is minor when compared to the major emission derived from the electronic excited state of the green fluorescent protein.
To account for the green emission, energy transfer was suggested as a possible mechanism [42,43] and the details of this process have subsequently been studied by Wampler and co-workers [43-45]. Although the isolated green fluorescent protein will not catalyze light production in the absence of luciferase it can change the color of the in vitro emission [43,44]. Fig. 9 illustrates the effect of adding the green fluorescent protein to an in vitro reaction containing luciferase and luciferin. The color of the light changes from blue to a characteristic green emission normally observed under in vivo conditions. In addition the quantum yield relative to luciferin increases from 5 ~ for the luciferase catalyzed reaction to about 25°~, in the presence of the green fluorescent protein. This is near the theoretical limit of 30% for the fluorescent quantum yield of the green fluorescent protein.
That portion of the bioluminescence emission which is green is dependent not only upon the presence of luciferase and the green fluorescent protein but upon their concentrations as well. The phenomenon is highly concentration dependent implying that an association of the two proteins is necessary prior to the emission of green luminescence [44]. Such an association could result in energy transfer between the electronic excited state of the oxyluciferin monoanion (IV in Fig. 4) and the green fluorescent chromophore as shown in Fig. 10. Since the overlap of the oxyluciferin
149
W~ve~oo ~(NM}
.......... Li . . . . . . . . . 400
~5 000
Fig. 9. Effect of addition of the green fluorescent protein (lower) on the luciferase-catalyzed in vitro bioluminescence of Renilla (upper).
Fig. 10. Pathway to both blue and green light emission in Renilla.
150
monoanion emission with the green fluorescent protein absorption is very good [43], and since this absorption is a strongly allowed transition, the conditions for non- radiative energy transfer are excellent. The sensitized fluorescence of the green fluorescent protein cannot be explained by F6rster-type energy transfer in homog- eneous solution [46]; the concentrations of green fluorescent protein used are several orders of magnitude too low to account for the observed transfer efficiency [43]. Therefore, we must invoke an association of green fluorescent protein with luciferase. Although there is no direct evidence for this, associated proteins Of these molecular sizes (approximately 24000 and 40000 daltons) could easily provide interaction distances within the 28 A critical distance calculated for this energy transfer model [43].
IIB-4. Control of the bioluminescentflash. It has been observed that light is produced when Ca 2÷ is added to crude extracts of Renilla prepared in the presence of metal chelators such as EDTA [47,48]. This type of calcium-induced bioluminescence is similar to that observed in the luminous jellyfish Aequorea by Shimomura and Johnson [2]. These investigators and their associates were able to demonstrate that a pure protein, with molecular weight of about 30000, can be isolated from extracts of Aequorea and that this protein produces a blue luminescence simply upon the addition of Ca 2÷ [2,49]. This calcium-induced luminescence was found to be independent of dissolved oxygen [2] and thus was most unusual in comparison to other biolumines- cent systems.
Hastings and Morin [40,41,47] noted that calcium-induced luminescence activity occurred in crude extracts of a number of species of bioluminescent Antho- zoans and Hydrozoans. They reported that this calcium-induced luminescence was oxygen independent although Harvey [12] had noted that luminescence in several Anthozoans was dependent on oxygen. They also made an analogy to Shimomura and Johnson's original work on photoproteins [2] and proposed that this whole class of luminescent proteins be referred to as calcium-activated photoproteins. In contrast Cormier and associates [50-52] demonstrated an oxygen requirement for the calcium-induced luminescence in Renilla and have since demonstrated this requirement in a number of bioluminescent Anthozoans examined (see Table I). In addition they confirmed the oxygen-independent reactions reported for the Hydro- zoans.
Clearly there is a difference between the calcium-induced bioluminescence of the Anthozoans and the Hydrozoans. The basic reason for this difference is the existence of a luciferin-binding protein in Renilla and other bioluminescent Antho- zoans (see Table I) which releases luciferin, specifically in the presence of Ca 2÷ [52]. Anderson et al. [52] have purified this protein completely free of luciferase and have studied some of its characteristics. From sephadex G-75 chromatography it has a molecular weight similar to that of luciferase, i.e. about 24000. It is a yellow-green protein with a visible absorbance maximum at 435 nm which corresponds with the visible absorption of luciferin in methanol [53]. In fact the yeUow-green color of the
151
protein was found to be due to bound luciferin [52]. The luciferin can be completely discharged from the binding protein in the presence of Ca 2÷. Subsequently the discharged binding protein can be recharged in the presence of synthetic or native
luciferin. Reconstitution of the calcium-induced bioluminescence in Renilla requires the
presence of charged binding protein, luciferase, oxygen and Ca ~+. No calcium- induced luminescence occurs in the absence of luciferase [52]. Since a luciferin- binding protein and Renilla-like luciferase have been observed in all Anthozoans examined (Table I), the participation of this calcium-triggered luciferin binding protein in the in vitro luminescence process in Anthozoans can be viewed as follows:
where BP refers to the luciferin binding protein. The oxygen requirement for this type of calcium-induced bioluminescence is now readily explained.
The discovery of this calcium-triggered luciferin binding protein in Renilla also provides an explanation for the old observation that Ca 2+ stimulates luminescence in extracts when initiated with luciferyl sulfate [17]. Discharged binding protein acts as an inhibitor of luciferase activity in the absence, but not in the presence, of Ca 2+. We also know that Ca 2÷ does not influence the activity of purified luciferin sulfokinase [92].
The calcium-induced release of luciferin from the binding protein apparently acts as a direct control mechanism for bioluminescence. Thus this places the luciferin sulfokinase reaction in a position to control the charging of the luciferin-binding protein.
A dramatic difference in kinetics is observed when one compares an in vivo flash to a calcium-induced in vitro flash (Fig. 11). Whereas the kinetics of the onset of luminescence are similar in each case the in vivo flash decays much more rapidly than the in vitro one [50,51]. Apparently in vivo bioluminescence is under very fine control. If the luciferin-binding protein is to play a key role in the control of bio- luminescence then a mechanism must exist in vivo for protecting this protein from Ca 2+. An obvious mechanism for stabilization of this protein would be to sequester it inside a membrane-enclosed particle. Such particles were demonstrated in extracts of Renilla and were found to produce green light (2m~ ~ = 509 rim) upon exposure to hypotonic solutions of calcium [50,51]. An analogous calcium-dependent partic- ulate bioluminescent system was also observed by Morin and Hastings [42] using extracts of the Hydrozoan Obelia. Anderson and Cormier [51 ] found that the particles from Renilla could be isolated as discrete vesicles in highly purified form. An electron micrograph of these vesicles is shown in Fig. 12. The vesicles are approximately 0.2 ~,m in diameter, are enclosed by a unit membrane, have an apparent density of 1.17 g- cm -3 in sucrose, and exhibit a fluorescence typical of the green fluorescent protein [51].
152
I i i
i E
SotgetE
TiME IN SK.
Fig. 11. Kinetics of the bioluminescence reactions of the soluble (in vitro), lumisomal and in vivo systems of Renilla.
Fig. 12. Electron micrograph of Renilla reniformis lumisomes.
153
When the vesicles are exposed to hypotonic solutions of C a 2+ they produce a flash of green light. Presumably the membrane ruptures allowing calcium access to the inside. The color of the light produced is identical to that observed from Renilla in vivo, i.e. identical to that shown in Fig. 8. Oxygen is also required for this calcium- induced luminescence. Anderson and Cormier [51] demonstrated that all of the proteins directly involved in Renilla bioluminescence and its control were present in these vesicles. These included the calcium-triggered luciferin-binding protein, luciferase and the green fluorescent protein. As yet luciferin sulfokinase activity has not been observed in preparations of these vesicles. These vesicles were termed lumisomes. The evidence indicated that all three proteins were tightly associated with the lumisome membrane. Only rigorous treatment such as high salt or sonication resulted in partial dissociation of any of these proteins from the membrane. The membrane association of these proteins within the lumisome is an important con- sideration in producing the observed green emission. Whereas under in vitro con- ditions such green emission apparently occurs due to protein-protein interaction, in vivo the lumisome membrane would provide an excellent environment for such interaction to occur.
The kinetics of the calcium-induced lumisome flash (Fig. 11) more nearly resemble the kinetics of the in vivo flash even though under these conditions the lumisome membrane is presumably ruptured. The decay of the lumisome flash can be made to exceed that of the in vivo one by rapid removal of calcium after initiation of the reaction [51]. This is analogous to the findings of Hastings et al. [54] who demonstrated the same phenomenon using the isolated photoprotein from Aequorea.
Thus the kinetics of the bioluminescent flash in Renilla can be accounted for by assuming that the lumisome containing cells (photocytes) exert careful control over calcium transients within the lumisome.
In other systems Ca 2÷ is known to play a direct role during the early phase of synaptic and neuromuscular transmission [55,56]. The calcium triggering of the luciferin-binding protein of Renilla and of the photoprotein of Aequorea is the logical choice of mechanism for linking the nerve impulse to the bioluminescent system. Thus the Renilla luciferin-binding protein, which can also be viewed as a calcium- binding protein, may serve as a model for events which occur during the calcium- dependent release of neurotransmitters by a nerve impulse.
IIC. Bioluminescence in other anthozoans A number of observations made over the past several years suggested that
similarities exist among the bioluminescent systems in coelenterates. The biochemical requirements for light emission of three species of Renilla (Table I) were found to be identical [57]. Cormier et al. [58] and Hori et al. [16] also reported that the components required for light emission in the Anthozoan Cavernularia obesa were identical to those found in Renilla. These included all the components listed in Table I except lumisomes and the luciferin-binding protein which were not yet discovered. Morin and Hastings [40-42] examined a large number of coelenterates
154
and found that extracts of all of them exhibited a calcium-induced bioluminescence. Furthermore, most of these organisms produce green light in vivo as opposed to the blue in vitro emission induced by calcium.
The above observations prompted Cormier and associates [45,51,52,59] to examine a large number of coelenterates in an effort to find out whether their chemical requirements for light emission were similar or identical to those already described for Renilla. Evidence was presented that all of the components required for bio- luminescence in Renilla as listed in Table I were found in all Anthozoans examined and that these components were indistinguishable. These observations extend to the manner in which the proteins involved in bioluminescence, and its control, are packaged. That is, particulate fractions with the physical and biochemical characteristics of lumisomes were found in the various Anthozoans as indicated in Table I. Thus the chemistry of light emission and the mechanism involved in the control of the bioluminescent flash as described for Renilla apparently apply to all the Anthozoans thus far examined.
III. BIOLUMINESCENCE IN THE HYDROZOANS
IliA. Aequorea and Halistaura (jellyfish) bioluminescence Approximately twelve years ago Shimomura et al. [2,60,61] isolated a most
interesting type of bioluminescent system from the luminous jelly fish Aequorea and Halistaura. When the luminous tissues were extracted with EDTA-containing buffers a pure protein component could be isolated. This protein exhibited a brilliant blue luminescence which decayed by first order kinetics, simply upon the addition of Ca 2+. The unique feature of this reaction was the observation that molecular oxygen was not a required reactant in agreement with an earlier observation by Harvey [62]. The isolated proteins were termed photoproteins. Specifically the one from Aequorea is referred to as aequorin and the one from Halistaura as halistaurin. Although the in vitro reaction produced blue light ('~max = 469 rim, corrected) visual observations suggested that the live animal produced green light. They also observed the presence of a green fluorescent protein in some of their fractions during their purification procedure. Cormier et al. [59] later isolated this protein from Aequorea and showed that its fluorescence emission was similar to that isolated from Renilla (see Fig. 8). Differences were noted in their fluorescence excitation spectra suggesting modifi- cations in either the chromaphore or the protein or both. The structured and narrow excitation band noted in Renilla [43] was broadened and blue shifted in Aequorea (unpublished). Measurements made by Morin and Hastings [42] confirmed that the in vivo luminescence of Aequorea was green and that this green emission was similar to that of the fluorescence of the green fluorescent protein reported by Johnson et al. [63]. Thus the production of green light in vivo most probably occurs by the same mechanism outlined for Renilla i.e. via energy transfer from an electronic excited state of the photoprotein reaction product to the green fluorescent protein.
Fig. 13. Absorption spectra of the photoprotein aequorin before luminescence (A), after lumines- cence in the presence of calcium (B) and of apoaequorin (C). Data taken from Shimomura and Johnson [64].
This possibility is enhanced with the finding by Anderson and Cormier [51] that the photoproteins and green fluorescent proteins from certain other Hydrozoans can be isolated packaged together in lumisome-like vesicles (see Table I) which represents an ideal environment for energy transfer. Unlike lumisomes isolated from Antho- zoans, the Hydrozoan vesicles do not contain a luciferin-binding protein.
The molecular weight of aequorin is approximately 23 000 by Sephadex chroma- tography and 31000 by sedimentation analysis [49,64,65]. The absorption spectrum of aequorin before and after the addition of calcium to initiate luminescence is il- lustrated in Fig. 13. Before light emission occurs aequorin exhibits a weak visible absorbance whose maximum was estimated to be 465 nm by Shimomura and Johnson [64] as shown in the inset to Fig. 13. More recent measurements by Cormier and associates (unpublished) have shown this absorption maximum to be 454 nm. When calcium is added light emission occurs which results in the disappearance of the 454-nm band and the appearance of a new absorption band at 335 nm with a ~'mM 335nm
of 13 [64]. During the process aequorin, which is not fluorescent, is converted to a blue fluorescent product referred to as the blue fluorescent protein. According to binding studies using ~SCa, 3 moles of calcium combine with 1 mole of aequorin to yield a protein-bound product whose fluorescence is identical to that of the bio- luminescence emission [66]. The bioluminescent quantum yield of aequorin is 0.23 at 25 °C and increases at lower temperatures [64,66].
Shimomura and Johnson [64] were able to remove a low molecular weight chromophore from the blue fluorescent protein. The resulting apoprotein has an absorption shown in Fig. 13 which demonstrates a loss of the 335-nm band. The 335-nm absorption was found to be due to the low molecular weight chromophore,
156
referred to as AF-350, which they isolated and initially identified as Compound II in Fig. 4 [67]. This structure was confirmed by chemical synthesis [68]. Note that AF-350 and Renilla etioluciferin are the same compounds.
Although Renilla etioluciferin (II, Fig. 4) was originally reported to be the light emitter in aequorin luminescence [67] more recent data shows it to be a breakdown product of the true emitter. Handling of the blue fluorescent protein in a different manner resulted in the isolation of the intact chromophore. Shimomura and Johnson recently deduced its structure and confirmed it by chemical synthesis [69]. Its struc- ture is shown as Compound V in Fig. 4. Note the near identity of this product with that of Renilla oxyluciferin (I, Fig. 4). For comparative purposes Compound V (Fig. 4) will be referred to as Aequorea oxyluciferin. Aequorea oxyluciferin (V, Fig. 4), like Renilla oxyluciferin (I, Fig. 4), is not fluorescent in an aqueous environment [69]. However, it has been possible to reconstitute the blue fluorescent protein by mixing together Aequorea oxyluciferin with apoaequorin and calcium. The fluorescence of the reconstituted blue fluorescent protein matches that of the aequorin biolumines- cence reaction [69]. Thus the emitter during aequorin bioluminescence appears to be a complex of Aequorea oxyluciferin and apoaequorin. This is analogous to the findings in Renilla in which a complex of luciferase and the oxyluciferin monoanion apparently represents the electronic excited state [14].
Based on the data outlined above and stopped-flow kinetic studies of the aequorin reaction with calcium [54,70,71 ] the reaction can be illustrated as follows:
aequorin q- Ca 2+ kt aequorin-Ca k2 X k3 BFP + light
where BFP refers to the blue fluorescent protein and X is a hypothetical intermediate. Values for the rate of binding of calcium to aequorin at 25 °C (k~) range from 7 • 106 M -1 ' s -1 to 1.2 • 107 M -1 • s -1 [66,70]. The rate of formation of blue fluorescent protein (k3) is two or more orders of magnitude greater than k2 [70,71] which is about 1 s -a. The large differences between the rate constants kz and k3 provide a ready explanation of why chelators such as EDTA inhibit the light reaction after its initiation [54,71 ]. Thus at the stage of calcium binding to aequorin the continual presence of free calcium is required for light production. This fact provides a good basis for the biological control of the bioluminescent flash since the cell need only monitor calcium flux to aequorin. This same principle probably applies to the calcium-triggered luciferin-binding proteins found in the Anthozoans since chelators inhibit the calcium-induced luminescence in an analogous manner [51 ].
As outlined above similarities of Aequorea bioluminescence to that of Renilla include near identities of the bioluminescent product (oxyluciferin) and the partici- pation of a green fluorescent protein whose fluorescence emission is similar to the one isolated from Renilla [59]. Another important similarity was discovered by Cormier et al. [59] who found that a Renilla-like luciferyl sulfate (IV, Fig. 2) could be isolated from Aequorea as well as from all of the bioluminescent Anthozoans examined. This Aequorea luciferyl sulfate was found to be identical to Renilla
157
luciferyl sulfate in its physical and chemical properties and in its interaction with Renilla luciferin sulfokinase.
IIIB. Comments on the nature of photoproteins As outlined above there are striking similarities between the bioluminescent
systems of Aequorea and Renilla. The appearance, after the aequorin luminescence reaction, of the 335-nm absorption band (Fig. 13) is now accounted for by the for- mation of Aequorea oxyluciferin [69]. Thus during both the Renilla luciferase- luciferin reaction and the aequorin--calcium reaction nearly identical products, Renilla and Aequorea oxyluciferins, are produced [14,69]. The formation of oxy- luciferin in each case is accompanied by the loss of visible absorption bands; the 435-nm band of Renilla luciferin [14] and the 454-nm band of aequorin [64]. In Renilla luciferin the emM 435mn in methanol is 9.7 [14] whereas in aequorin the emM 454m" is only 2 [69]. This has been the primary argument [67,69] against there being a chromophore in aequorin with a fused imidazole ring such as that found in Renilla luciferin (II in Fig. 2). This is not a good argument since the protein could conceivably induce environmental effects, coupled with protein-chromophore interactions, re- suiting in absorption shifts and changes in molar absorptivities. In fact we have recently demonstrated that the absorption spectrum of Renilla luciferin, under appropriate environmental conditions, can be made to mimic the long wavelength absorption of aequorin (Hori et al., unpublished). Under such conditions the 435-nm absorption band of luciferin shifts to 454 nm with a concomitant lowering of the visible absorption emM from 9.7 (435 nm) to about 2.0 (454 nm). Another reason given for the assumed lack of a fused ring is the observation that dissolved oxygen is not required for the aequorin luminescent reaction [2,3]. As illustrated in Fig. 7 for Renilla bioluminescence oxygen attacks the fused ring early in the reaction but forms a hypothetical luciferin hydroperoxide intermediate. Thus during the for- mation of aequorin a Renilla-like luciferin, derived from a Renilla-like luciferyl sulfate, could be incorporated into the protein and stabilized in the absence of calcium, as a protein-luciferin hydroperoxide intermediate. A stabilized oxygenated inter- mediate such as a peroxide has been suggested by several groups [4,8,69,72-75]. This would account for the lack of an oxygen requirement. Furthermore it would help satisfy the energy requirements necessary for the creation of an electronic excited state and the scheme would now be consistent with theories on the oxidative mechanisms involved in bioluminescence [4]. The lack of CO2 production during the aequorin luminescent reaction [76] could be accounted for by assuming a covalent linkage between a group on the protein and the carbon from which CO2 is normally derived in these reactions.
From the above discussion it appears that there is no basic difference in the chemical path to light emission in Aequorea and Renilla. The basic difference is at the protein level and this reflects differences in how the luminescence is controlled in each case. Fig. 14 illustrates the differences and similarities that exist between the Hydrozoan and Anthozoan luminescent systems. Note that, in the Anthozoans, two
158
PATHWAY TO BIOLUMINESCENCE IN THE ANTHOZOANS:
Binding Protein-Luciferin + C a + + ~ Binding Protein-Ca + Luciferin
l 1 Luciferase-Oxyluciferin + Light (Blue) GFP + Light (Green)
Photoprotein + Ca ++
Photoprotein-Ca
Protein-Oxyluciferin* + GFP
1 Protein-Oxyluciferin + Light (Blue)
PATHWAY TO BIOLUMINESCENCE IN THE HYDROZOANS:
Photoprotein-Ca
> Protein-Oxyluciferin* (Blue Fluorescent Protein
GFP* + Protein-Oxyluciferin
GFP + Light (Green)
Fig. 14. A comparison of the bioluminescence pathways in the Anthozoans and the Hydrozoans.
proteins are involved in the calcium induced production of blue bioluminescence in contrast to the Hydrozoans where only a single protein is involved. In the case of the Hydrozoans it is apparent that the point of oxygen entry occurs prior to the formation of the photoprotein. The photoproteins are viewed as playing a dual role. They apparently bind a Renilla-like luciferin hydroperoxide thus acting like a binding protein and they allow conversion of this luciferin hydroperoxide in the presence of Ca 2+ to a Renilla-like oxyluciferin thus also acting like a luciferase.
IIIC. Bioluminescence in other hydrozoans Extracts prepared from a number of Hydrozoans (see Table I) contain photo-
proteins with properties similar to those described above for Aequorea [40-42,59]. All those examined contain a green fluorescent protein similar to the one described for Renilla [41,43]. However, we have noted minor spectral shifts in the in vivo bioluminescence emissions of several Hydrozoans such as Obelia and Clytia when compared to Anthozoans such as Renilla (unpublished). These same spectral shifts are also observed when the fluorescence of the isolated green fluorescent proteins from Obelia and Clytia are examined. Certain Hydrozoans (Table I) have also been found to contain a Renilla-like luciferyl sulfate [59] and package the proteins involved in their bioluminescence into lumisome-like vesicles [51].
159
IV. BIOLUMINESCENCE IN THE CTENOPHORES
1VA. Biolumineseence in Mnemiopsis and Bero~ (comb jellies) Some important observations on these rather transparent and brilliantly
luminescent creatures were made over a century ago. Whole animal experiments by Allman in 1862 [77] demonstrated that visible light inhibited their ability to luminesce and that this inhibition was reversed by a few minutes in the dark, suggesting a control mechanism for bioluminescence based on photosensitivity. He also observed that the early segmentation stages of the developing embryo of Mnemiopsis leidyi were luminous. An extensive investigation of the development of bioluminescence during ernbryogenesis in Mnemiopsis leidyi has been conducted recently by Freeman and Reynolds [78]. By use of modern cytological and image intensification techniques they were able to describe the location and certain properties of the photocytes in adult animals. They were also able to describe the stage at which the development of bioluminescence occurs, the location of the photocytes at that stage and at subsequent stages of development from embryo to larvae.
Another important observation on whole animals was made by Harvey [79] who demonstrated that luminescence occurred in the absence of oxygen. More recently Morin and Hastings [41,42] demonstrated that extracts of Mnemiopsis leidyi can be prepared which produce a flash of light upon the addition of Ca 2+. They also observed that the luminescent reaction was oxygen independent and thus the activity in the protein fraction was classified as a photoprotein because of its simi- larities to the Hydrozoan photoproteins described above.
A distinct difference between the Hydrozoan and Ctenophore bioluminescence systems was noted by Morin and Hastings [41] who found that the in vitro and in vivo bioluminescence emissions were similar. A more careful examination of the bioluminescence emission characteristics of two species of ctenophores by Ward and Seliger [80] does show a slight red shift in their in vivo emissions (see Table I). A lack of green fluorescence in the region of the luminescent tissues [42] suggested the absence of a green fluorescent protein analogous to those observed in the Hydrozoans [42,45,59].
Ward and Seliger [81 ] were the first to report the purification to homogeneity of two soluble photoproteins from a ctenophore, Mnemiopsis sp. which they referred to as m-1 and m-2. They also reported the purification of a photoprotein from the ctenophore Bero~ ovata [82] and during the same year Girsh and Hastings [83] reported the purification of a similar photoprotein from Mnemiopsis leidyi. The photoproteins isolated from Mnemiopsis have been termed mnemiopsin [83,84] while the one isolated from Bero9 has been termed berovin [84] in keeping with previous nomenclature [2]. These photoproteins produce an oxygen-independent lumines- cence in the presence of Ca 2+ and thus are analogous to those isolated from the Hydrozoans.
Mnemiopsins m-1 and m-2 occur in multiple ionic forms and by polyacrylamide gel electrophoresis in sodium dodecylsulfate, are single polypeptide chains of molec-
160
.06 ABSORPTION SPECTRA OF NATIVE
AND REACTED M-2 PHOTOPROTEINS
~.o4
o_
~,o2
. O 0
m
m
B ~ A . I l l I IJI
C .
I JlllllliJlJlllll I l l l l l l l l l l 2~ 3000 ~fc)O0 4000 4500 5000 5500
WAVELENGTH ,~ Fig. 15. Absorption spectra of the photoprotein mnemiopsm (m-2) before luminescence (A) and after luminescence in the presence of Ca z+ (B). Curve C is the baseline. Data taken from Ward and Seliger [84].
ular weights 24000 and 27500 [80]. Berovin was found to be a single polypeptide chain of molecular weight 25000 [80] using the same technique. The reader is referred to the papers of Ward and Seliger [80,84] for information on the general properties, kinetics, emission spectra and quantum yields of mnemiopsin and berovin.
Whereas aequorin shows a visible absorption maximum at 454 nm [64] mnemiopsin exhibits one at 435 nm [80] as illustrated in Fig. 15. As with aequorin when luminescence is induced by calcium addition the visible absorption band disappears followed by the appearance of a new band at 335 nm (Fig. 15). This is reminiscent of events which occur during the reaction of aequorin with calcium as outlined in Section III above. Unlike aequorin, however, the product of the mnemi- opsin reaction was found not to be fluorescent [80].
As was observed with whole animals Morin and Hastings [41 ] noted that visible light rapidly inactivated the calcium-induced luminescence of mnemiopsin. A careful study of this phenomenon by Ward and Seliger [82,85] revealed several interesting observations. Photoinactivation was found to occur over the entire region of its absorption spectrum, i.e., from 230 to 570 nm. The quantum yield of inactivation at 2537 A of 0.026 makes mnemiopsin one of the most light sensitive protein-chromo- phore complexes known [85]. Above 400 nm the photoinactivation quantum yield was found to be independent of wavelength suggesting that absorption by the chromo-
161
phore must be solely responsible for photoinactivation in the visible region. The photoinactivation action spectrum of mnemiopsin is similar to its ab-
sorption spectrum. The visible absorption at 435 nm is very similar to the absorption of Renilla luciferin in methanol [53] or to that of the Renilla tuciferin-binding protein [52]. Furthermore the loss of this 435-nm band during luminescence, followed by the appearance of the 335-nm absorption, has striking similarities to absorption changes noted during the bioluminescent oxidation of Renilla luciferin by luciferase [14]. Thus it has been proposed [80,85] that ctenophore photoproteins also contain a Renilla-like luciferin chromophore which, after luminescence in the presence of calcium, leads to the formation ofa Renilla-like oxyluciferin product. In this regard it is of inter- est that Cormier et al. [59] have isolated a Renilla like luciferyl sulfate from Mnemiopsis.
Photoinactivation of mnemiopsin appears to involve an irreversible disruption of a Renilla-like luciferin chromophore in the protein [85]. During this process an oxyluciferin-like product is not formed.
V. APPLICATIONS OF COELENTERATE BIOLUMINESCENCE
In recent years the applications of bioluminescence have become widespread for a number of reasons. Firstly, a number of the luciferases, luciferins and photo- proteins have been isolated and several of the luciferins have been synthesized. Secondly, modern electronics have provided highly sensitive and convenient devices for detecting light. Thirdly, the measurement of light production is an extremely sensitive probe for the specific detection of many biologically important compounds. It is not unusual to detect specifically 10 -15 mole of a compound or even less depend- ding on the available instrumentation. For a summary of the major applications of bioluminescence the reader is referred to a review by Seliger [86].
In 1963 Shimomura et al. [87] described a rapid micro method for the detection of Ca 2÷ based on the specificity of the aequorin bioluminescence for calcium. Sensitiv- ities on the order of 10 -2 ~g of calcium per ml were described.
Ca 2+ has been proposed to play important roles in biological processes such as excitation-contraction coupling in muscle and synaptic and neuromuscular trans- mission. Thus aequorin bioluminescence has been utilized as a probe in following the rapid calcium transients which occur during these events. For example microinjection of aequorin into the presynaptic terminal of the giant squid synapse demonstrated an increase in intracellular Ca 2+ concentration during repetitive synaptic transmission [88]. This increase was found to be associated with repetitive activation of the presynaptic terminal suggesting that a flux of Ca 2÷ into the axoplasm occurred. Aequorin has also been used to follow calcium-uptake in isolated mitochondria [89].
In experiments on excitation-contraction coupling, single muscle fibres, injected with aequorin, were found to emit light when stimulated to contract [90,91]. These investigators were able to record simultaneously changes in membrane potential, calcium transient and tension during a single contraction. They were able to directly
162
demonstrate a transient increase in the sarcoplasmic calcium concentration during the rising phase of tension. Kinet ics o f the b ioluminescence of aequor in are ap-
paren t ly sufficiently r ap id to re f lec t t rue changes in ca lc ium transients in these, but not
all, such exper iments [54].
Because of the react ions involved in p roduc ing light by Renilla (Section II) it
is clear tha t very sensitive assays can be deve loped for 3 ' , 5 ' -d iphosphoadenos ine ,
adenylyl sulfate 3 ' -phosphate , and enzymes involved in their ut i l izat ion and synthesis
[15,17,93].
Vl. ACKNOWLEDGEMENTS
The au thors are grateful for use of facilities p rov ided by the Univers i ty o f
Geo rg i a Mar ine Inst i tute , Sapelo Is land, Georg ia , U.S.A. and for research suppor t
by the N a t i o n a l Science F o u n d a t i o n (GB-28559X) and the U.S. A tomic Energy Com-
mission (AT (38-1)-635).
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