THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 248, No. 8, Issue of April 25, pp. 2937-2943, 1973

Printed in U.S.A.

Lumisomes, the Cellular Site of Bioluminescence

in Coelenterates*

(Received for publication, November 7, 1972)

JAMES RI. ANDERSON AND MILTON J. CORMIER~

From the Department of Biochemistry, University of Georgia, Athens, Georgia 30601

SUMMARY

From ten species of bioluminescent coelenterates we have isolated a membrane-enclosed subcellular particle that is apparently responsible for the bioluminescence in these animals. The animals investigated were species of Obelia

and Clyfia (from the class Hydrozoa) and species of Renilta,

Pfilosarcus, Sfylafula, Acanfhopfilum, and Parazoanfhus

(from the class Anthozoa). By electron microscopy the purified particle preparations appear quite homogeneous in size and shape, have a diameter of approximately 0.2 pm, and are enclosed by a unit membrane. The particles con- tain all of the proteins necessary for producing the typical green bioluminescence (h,,, 509 nm) observed in vivo in these animals. These include luciferase, a calcium-triggered photoprotein and a green fluorescent protein. Biolumines- cence is evoked from the particles when they are placed in hypotonic solutions of calcium ion. Oxygen is also required for light emission. The color of the light is green, typical of the observed in vivo emissions. The widespread occurrence of these bioluminescent particles among the coelenterates suggest that they represent the cellular site of biolumines- cence in these animals. We propose that these subcellular bioluminescent particles or organelles be referred to as lumisomes.

In recent years there has been considerable progress made on the biochemistry of bioluminescence among a diverse group of marine animals known as the coelenterates. Two classes of coe- lenterates that have been intensively studied within the Phylum Cnidaria are the Hydrozoa and the Anthozoa. These studies have followed two paths. One of t,hese has centered on a soluble protein isolated from the Hydrozoa that produces light upon the addition of calcium ions (l-6). Such proteins have been termed phot,oproteins with the reaction shown in Equation 1.

Photoprotein + Ca+* + hv (hmax- 460 nm) (1)

* This work was supported by grants from the National Science Foundation and the United States Atomic Energy Commission. This is Contribution 236 from the University of Georgia Marine Institute, Sapelo Island, Georgia.

f Career Development Awardee I-K3-6M-33-07 of the United States Public Health Service.

Another pathway of study has centered on a series of enzymes isolated from the Anthozoa (7-12). These enzymes catalyze the reactions outlined below:

Luciferyl sulfate + 3,5’-diphosphoadenosine (DPA)

luciferin sulfokinase ’ luciferin (2)

+ 3’-phosphoadenylyl sulfate (PAPS)

luciferase Luciferin + 02 w oxyluciferin* + CO, (3)

Oxyluciferin* + oxyluciferin + hv (X,,, N 490 nm) (4)

With the discovery that protein preparations which produce light in the presence of calcium, similar to photoproteins, exist in all bioluminescent Anthozoa examined (13-15), it seemed possible that coelenterates possess a common biochemical mech- anism for bioluminescence and that both areas of study outlined above were parts of the same problem. Recent evidence pro- vides additional support for this proposal (16). For example, Renilla-like luciferyl sulfate has been found in eight species of bioluminescent coelenterates, including certain Hydrozoa, which cross-reacts to produce light with anthozoan luciferin sulfokinase and luciferase as illustrated by Equations 2 to 4. Furthermore Renilla-like luciferases have been found in eight species of an- thozoans while Renilla-like luciferin sulfokinases have been de- tected in seven species of anthozoans (16).

The in vitro reactions described above all produce blue light onlax - 460 to 490 nm) which shows a broad spectral energy distribution. This is in contrast to the in viva emission of most

coelenterates which is green (X,,, = 509 nm) and exhibits a narrow spectral energy distribution with vibrational structure at 540 nm (10, 15). The green in vivo emission has been postu- lated to be due to energy transfer from the electronically excited state of oxyluciferin to a second protein-bound chromophore (10, 14, 17). This protein-bound chromophore exhibits fluores- cence characteristics identical with the in viva bioluminescence. It has been isolated from extracts of Renilla renijormis (10, 26) and is referred to as the green fluorescent protein. Energy transfer between the two chromophores apparently requires protein-protein interaction between the green fluorescent pro- tein and luciferase (26).

Photoproteins are apparently an important physiological entity in controlling the bioluminescent flash normally observed in coelenterates. In order to stabilize photoproteins they must be protected from cellular calcium. An obvious mechanism for

2937

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intracellular stabilization of photoproteins would be to sequester them inside a membrane-enclosed particle. Such a particle would also be espected to contain a green fluorescent protein in those cases where green in viva bioluminescence is observed. Morin and Hastings (17) presented evidence that such a particle esists in Obelia, a hydrozoan. A crude preparation of a particu- late-like photoprotein activity was obtained by sedimentation that apparently required lysis of the particle, as well as the presence of calcium ions, prior to light emission. Although no spectral data was presented they indicated that the light emitted was green, typical of the in vivo bioluminescence.

The observations outlined above prompted us to look for the existence of bioluminescent particles in Renilla and other coe- lenterates. A study of such particles could provide insight into the excitatory mechanisms controlling the bioluminescent flash in vivo. We present evidence in this paper that photoproteins in coelenterates are indeed packaged within membrane-enclosed particles which can be isolated as discrete vesicles. We also report t.hat such particles contain all of the necessary proteins required for producing the typical green bioluminescence ob- served in many coelenterates in vivo.

We propose that these subcellular bioluminescent particles or organelles described here be referred to as lumisomes.

METHODS

Renilla miilleri were obtained from Gulf Specimen Company, Inc., Panacea, Fla. Renilla reniformis were obtained by collec- tion at Sapelo Island, Ga. Obelia geniculutu were obtained from the Marine Hiological Laboratory, Woods Hole, Mass. All other organisms were gifts of Dr. James Morin, University of California at Los Angeles. They were maintained in circulat- ing artificial sea water (Instant Ocean).

Emission spectra and fiuorescence characteristics of lumisomes were determined by using a component fluorometer linked ow

line to a Nova computer (18) as described previously (10). Kinetic studies were done using a manual stopped flow apparatus linked online to the Nova computer (19).

Electron microscopy of lumisomes was carried out by the Electron Microscopy Laboratory of the T:niversity of Georgia. Lumisome samples were fixed in 2% gluteraldehyde in the ho- mogenizing medium for 2 hours at 4”. Samples were washed in three changes of the homogenizing medium and post fixed in 2% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.2, for 2 hours at 4’. Preparations were dehydrated in a graded series of ethanol followed by embedding in Marsglass. Electron mi- crographs were taken with a Phillips EM 200 at 80 kv.

Assay of Proteins Associated with Bioluminescence-Soluble proteins were separated from lumisome-associated proteins by centrifugation at 20,000 x g. Luciferase activity was assayed by adding 0.2 ml of luciferin to a 0.1.ml sample in 1.0 ml of 0.1 M potassium phosphate buffer, pH 7.5, containing 0.01 in EGTA’ as describecl previously (9). Lumisomes were lysed by injection into the phosphate buffer. Sulfokinase activity was assayed as described previously (8). The green fluorescent protein was extracted from purified lumisomes by first adding lumisomes to saturated ammonium sulfate. The precipitate, collected by centrifugation, was dissolved in 0.1 RI potassium phosphate buffer, pH 7.5, and the particulate material removed by cen- trifugat.ion. The fluorescence of the supernatant was det,ermined as previously described (10). Soluble photoprotein was assayed by adding 0.2 ml of 0.05 &r CaC12, pH 8.5, in 0.6 1\1 NaCl (isotonic)

1 The abbreviation used is: PITA, ethylene glycol bis(amino- ethyl ether)-N,X’-tetraacetic acid.

to 0.1 ml of the sample. The final concentration of calcium required to initiate the reaction was set by the concentration of EGTA. This was verified down to 1 X lo-” M CaCl* and EGTA. Lumisome-associated photoprotein was assayed by adding 0.6 ml of water to the above described soluble photoprotein assay mixture or 0.4 ml of 0.01 M CaC&, pH 8.5, to 0.1 ml of the sample.

Preparation of Lumisomes-The animals were soaked for 15 to 30 min in 600 mM NaCl containing Na2EGTA, 20 mM, to remove soluble calcium. They were then placed in the homog- enizing medium (NaCl, 500 mM; MgCla, 1.25 mM; Na2H1’04, 20 mM, pH 7.8; Na*EGTA, 20 mM) and cooled to 4”. ,4 ratio of about 10 g of animals to 40 ml of buffer was used. The tissue was first chopped with a pair of scissors and then homogenized with a Willems Polytron I’T 20st set at full speed for 15 s. The resulting brei was centrifuged at 270 x g for 20 min and the supernatant filtered through Miracloth. The fraction which sedimented between 1,000 and 20,000 X g during a 20.min cen- trifugation was used as crude lumisomes. Lumisomes were fractionated in slightly concave 0.5 to 1.5 M sucrose density gradients which were formed using an ISCO programmed gra- dient pump. The sucrose solutions contained the same salts as the homogenizing medium. Gradients of 11.5 ml were centri- fuged at 148,000 X g for 12 hours (Spinco SW 41 Ti) at 5”. They were then fractionated into 0.6.ml samples and the sucrose re- moved from the particles by dialysis since the particles were sensitive to sudden osmotic changes, A second sucrose gradient was used for further purification of lumisomes as described in Fig. 3.

RESULTS APr’U UISCUSSION

Characteristics of Soluble and Particulate Photoprotein from Renilla-l’hotoprotein activity extracted from Renillu was found to be of two types: a soluble fraction which, in similarity to other coelenterate photoproteins, produced blue light (X,,, -490 nm) on the addition of calcium ions; and a particulate fraction which could be sedimented from Renilla extracts at 1,000 to 20,000 X g. The particulate fraction produced green light (X,,, = 509 nm) which exhibited a narrow spectral energy distribution (Fig. 1).

Due to the noise level of some of the spectra in Fig. 1, all of the spectra were left uncorrected. Hecause of this the vibrational structure normally observed at 540 nm is not obvious. In the case of Renillu the noise level was sufficiently low that corrected spectra, clearly showing this vibrat,ional structure, were ob- tainable. Thus the color of bioluminescence of the particulate fraction was typical of the in vivo Renillu light emission while the soluble fraction emission was identical with the in vitro luciferase-luciferin reaction (10). As observed with the particu- late material isolated from Obeliu by Morin and Hastings (17), the particulate photoprotein of Renillu required both a drop in ionic strength and the presence of calcium ions to produce light. The particles were apparently osmotically lysed by the change in ionic strength. Furthermore the particles could also be dis- rupted by other mechanisms such as sonication. Light could then be produced by additions of isotonic calcium. The data suggest, in agreement with the work of Morin and Hastings (17), that photoprotein is encased in a membrane-bound vesicle. We suggest that these light-producing vesicles be referred to as lumisomes.

Isolation and Characterization of Lumisomes-Lumisomes were difficult to separate into discrete size classes by direct sedimenta- tion due to a slimy material which cosedimented between 2500 and 5000 x g. They could, however, be separated from the slimy material by banding on sucrose density gradients (Fig. 2).

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Twelve hours of centrifugation were required to remove the slimy materials from the lumisome banding regions of the gra- dient. Two major bands of lumisome activity were found at density regions of 1.17 and 1.13 g cmW3 and a minor band at 1.10 g cmV3 (Fig. 2). I f lumisome activity from any region of the sucrose gradient was subjected to a second sucrose gradient those lumisomes rebanded at density 1.17 g cme3 (Fig. 3). Con- taminating particles rebanded at their original density regions as shown by the ultraviolet absorption pattern in Fig. 3. Thus, a

WAVELENGTH (nm) IO0 500 600 500

S. ELONGAT

7 I’,

00 2000

P.GUERNYI 1

R.MULLERI

A&!!!!! 1

00 2000

L

WAVE NUMBER (cm-l) FIG. 1. The emission spectra of the if& vitro photoprotein reac-

tion of lumisomes. Spectra are an average of several scans using the spectrofluorimeter described under “Methods.” These spec- tra are uncorrected for photomultiplier response.

Density g cm-3 J1.17

10.

I - 1 6 11 16 22

FRACTION

J’ FIG. 2. Distribution of particulate photoprotein activity

(0-O) and other ultraviolet absorbing material (O- --0) from Renilla mdleri on sucrose density gradients. Measurement of equilibrium density position of lumisome bands in the gradient was confirmed on several other gradients. A absorbance (280 nm to 290 nm) was used to reduce effects of light scattering from par- ticles on the 280 nm absorbance. 8 The band of ultraviolet absorb- ing material denser than 1.17 g cm-3 was bright orange and was absent from preparations from Renilla reniformis.

2939

considerable degree of purification could be obtained by taking lumisome material from the 1.10 g crne3 density region of the first gradient and rebanding it on a second gradient (Fig. 3A).

The shift in apparent densities to 1.17 g crnm3 during the second sucrose density gradient run may be associated with alterations in membrane structure during handling. For example we noted that storage of lumisomes, as well as subjecting them to sucrose gradients, apparently resulted in them becoming leaky to cal- cium ions since an increase in background luminescence occurred with such preparations when exposed to isotonic calcium (see Fig. 8C). This background luminescence was usually less than 10% as intense as the luminescence from lysed particles (Fig. 80). In this connection the particles from the 1.13 g cmP3 density region were less leaky than those from the 1.17 g cmP3 region indicating that the density change has to do with alterations in the membrane and possibly the amount of sucrose uptake.

Lumisomes purified by rebanding Fraction A of Fig. 3 on a second sucrose gradient were used for electron microscopy. As shown in Fig. 4 the purification procedure resulted in a fairly uniform preparation of vesicles, approximately 0.2 pm in diam- eter, which were enclosed by a unit membrane.

Metal Ion and pH Requirements for Lumisomal Activity- Renilla lumisome photoprotein specifically requires calcium ions for maximal activity. As shown in Table I essentially no lu- minescence was observed upon lysis in distilled water. Further it is the cation, not the anion, which is active. This require- ment for calcium ions is similar to that observed with the soluble photoprotein from Renilla. Several other divalent ions, how- ever, also evoked significant levels of luminescence (Table I).

1 6 11 16 21 FRACTION

I

16 FRA,:‘,Otl

16 21

FIG. 3. Distribution of lumisomes on sucrose density gradients. Lumisomes were isolated from Renilla renijormis followed by fractionation on an initial sucrose density gradient (i). The fractions from each of four regions (A, B, C, D) were pooled sep- arately and dialyzed to remove sucrose. Particulate material was removed and fractionated on a second sucrose gradient. A, B, C, and D, therefore, are the refractionation of lumisomes in regions A, B, C, and D, respectively, of the original gradient (i). Lumisome activity (O--O) and absorbance at 280 nm (a- - -0) are shown. The arrow denotes position of density 1.17 g cm-3 in each gradient.

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FIG. 4. ItXectron micrograph of Eenillu renijormis lumisomes. Lumisomrs were purified on two sucrose gradients as indicated in Fig. 3.

T.\IH,rr I

IOU speciJicil!/ of IL’etiilla miilleri lumisowles ~.--- ____I_ ~. ..~

Ion ndrleY : Calcium-stimulated light emkid .- ._____ --.. ~ .._~.-.- ~_..

‘;/o (‘.p 4. /‘ 100 Sr. ./. 26

13X”+ I 1 Co’-+ 22

CU*- 21

ivn++ 31 Zn”

j 20

p(d-+ 21

I‘ll++ 6

Pb++ 2

Mg++ I <l

NH,* <I Ii’ <1 NOM! <I

~1 All ions were 0.8 rnM final concentration with a 0.2 mM final concentration of lC(+TA.

* Percentage of ralcium-stimnlatcd quanta per s at peak in- tensity (2.3 X IO9 hv s-l).

One possible explanation for the partial activities observed with somr of these other ions may have t,o do with the fact that most divnlcllt ions have a higher bintling affinity for EGT,Z than pal- cium with t’lic esccptions of thr ions of magnesium, barium, and strontium (20). Thus contaminating calcium ions bound to EGT;\ might be r&aced upon the addition of ions with higher stability constants. The apparent activity, thercforc, of dica- lent ions other than calcium, strontium, and barium could lx a result, of compctit,ion for binding sites of ICGTX.

When the final concentration of magncsiuin in our phot.oprotein :~:ays XIS sufficiently high (0.1 x1) t,his ion fuuct.ioned as an effect,& competitive inhibitor of calcium. This was true both for the ~olublc and the particulate photoprotein assqs. Similar inhibitions of photoprotein activity by magnesium has been ob- scr\-cd using soluble photoproteins isolated from Obelia, Jlnemi- opsis, :UK~ Aequorea (15, 21).

Renilla lumisome photoprotein, therefore, behaves in a similar manucr to that of a number of soluble photoproteins previousl> described iu its divalent ion requirements (2, 3, 15, 22).

.a -

I t Argon I * I

0 15 30 45 MIN

FIG. 5. Dependence on oxygen of calcium-induced luminescence of lllmisomes isolated from 12e)tiZla renijormis. Lumisomes, sus- pended in 2.0 ml of homogenizing medium (see “Methods”), and 6 ml of 0.01 M calcium chloride were made anaerobic separately by passing argon through the solutions for 30 min. The solutions were mixed (Ca) causing lysis of the lumisomes and some light production. hfter 3 min, oxygen was passed through the solution (02).

The pH profile for Renilla lumisomnl and soluble photoprotein a(*tivity was similar to that, reported for other soluble photo- protein:: (2, 15). As in the other cax~ the Renilla photoproteins were most active when assayed near pII 9.0.

Oxygen Requirement for Light Emission jrom Lumisomes and Soluble Photoprolein-It has been found t.hat, the calcium-trig- gered photoproteins isolated from Obelia longissima, Aequorea forskalea, Pelagai noctiluca, Xnemiopsis leidyi, and Renilla kijllikeri represent os~gea-indcl)elldellt bioluminescent reactions (2, 15). 111 the case of Renilla renijormis WC know from previous studies (23) that the Renilla luciferin-luciferape reaction shoxn in Equation 2 is os~geil-depe~lde~~t. 13;~ carefully reducing the dissolved oxygen concentration to a sufficiently low level, we find that the calcium-triggcrcd luminescence derived from puri- fied Renilla lumisomes (Renilla ren$ormis and Renilla miilleri) is also osv~en-del,endeilt. These results arc presented in Fig. 5. Similar rcjults wcrc obtained using crude lumisomal prepara- tions. The dcpcndencc was not complete, however, as contam- inating oxygen iu the argon ( <5 ppm) cawed some light to be produced when non-isotonic calcium was injected under an argon atmosphere. Introduction of oxygen, subsequent to calcium addition, produced a large increase in light intensity. Similar results to these have also been observed by us using the calcium- triggered soluble photoprotein isolated from Renilla renijormis.

The observation that both the soluble and particulate photo- protein activities in Renilla were oxygen-dependent. reactions prompted us to look at whole animal luminescence to determine whet,her or not such osygeu dependence also occurs in vivo it1 Renilla.

Fig. 6 shows that argon inhibition of in vivo Renilla biolu- minescence is observed aud that this inhibition is reversed by osygeu.

Thus an oxygen dependcncc for Renilla bioluminescence can be observed using uot, only the components of the in vitro re- action, i.e. lucifcrin and luciferase, but also using the soluble

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HOURS

FIG. 6. l+Xfect of anaerobiosis on Renilla reniformis biolumines- cence. The organism was placed in distilled water to induce bioluminescence. Bioluminescence induced in this way appears to remain intracellular and the color of the light is green. Argon and oxygen were bubbled through the water at the points indi- rnt,ed.

photoprotein, the particulate photoprotein (lumisomes), and the whole animal. The differences in the osygeu requirements for Renilla renijormis bioluminescence and those coelenterates mentioned above is not easily understood. Two possible es- planations come to mind; either the oxygen-independent photo- proteins contain a stabilized hydroperoxide intermediate of some sort’ (24) or the concentration of dissolved oxygen required to saturate is much lower than in the case of Renilla renijormis making the requirement difficult to demonstrate.

Composition of Lumisomes--After centrifugation, the super- natant from a crude lumisome preparation contains all of t,he detectable luciferill sulfokinase, more than 90%, of the total luciferase and less than 90/c of the total detectable photoprotein activity. The remxitling quantities of luciferase aud photo- protein arc located withill the lumisome. In fact it is possible, although difficult, to extract from purified lumisomes the follom- ing proteins: luciferase, a calcium-triggered soluble photoprotein, and the green fluorescent protein (see ‘Xethods”). The fluores- cence of the green fluorescent protein extracted from lumisomes was identical with that of a green fluorescent protein previoublg isolated from Renilla (10, 26). These proteins also slowly solu- bilize frorn lumisomes during storage. The soluble proteins found in the supernatant described above could, therefore, repre- sent either solubilizatioll products from lumisomes, a precursor pool for new lumisomes, or a combination of both.

Lucifcrase, photoprotein, and the green fluorescent protein appear to be membrane-bound within the lumisome. Part of the evidence for this statement comes from the observation that these ljroteills are not easily solubilized from a lumisome prep- aratioll either by sonication or by treatment with 2.5 M NaCl. Furthermore when lumisomes are lysed with distilled water and the particles; collected by centrifugation it is found that esperl- tially all the calcium-triggered luminescent activity is recovered in the re;uspended pellet. In addition lumisornes appear as green fluorescent particles when viewed with a Zeiss fluorescence microscope. Thiq observation is consistent with our finding that lumisome< colltain the green fluorescent protein. Finally Table II showvs that we observe a green bioluminescence emis- sion (A,,,,, = 509 nm) from lysed lumisomes after various times

T:\HLE II Va,riatioris i/h dislr&ulion of bioluminescence belween green and

blue emission components for lumisomes

Lumisomes isolated from

Plilosarcus guernyi

Stylolule elongala

Retklla mdleri

Obelia ger~iculala

-I-

Addition times for Relative ratioa Ca+- after lysis (green)/blue

s

0

90

0

90

0

60 300

0 5

15 60

32 2s 66 47 14 14 19

1.8 1.7 2.2 1.4

= Data obtained from comparing spectra of bioluminescent flash as in Fig. 1 except Obelia geniculata where total light passing through variable wedge interference filter (Oriel Optics Co.) was compared at 509 nm and 470 nm.

0 SECdNDS

2

FIG. 7. Kinetics of in vivo bioluminescence of Reklla miilleri. Arrow denotes point of mechanical stimulation. The curoe repre- sents a tracing from the oscilloscope readout, of the Nova Mini- computer (19).

of lysis and up to 5 min in the ca:e of Renilla. Thus the green fluorescent protein does not dissociate from the lumisomal mem- brane under theFe conditions. Table II also shows that the same observations are made when lumisomes are prepared from Ptilosarcus, Stylatula, and Obelia.

Observations on Kinetics of Light Emission-When rr,echan- tally stimulated, Renilla produces a luminous flash whose kinetics is showli in Fig. 7. In this ca:e we are viewing a relected tissue area, thus the luminescence rike and decay times may not repre- sent minimal ones. Lumisomes also produce a luminous flash upon lysis in non-isotonic calcium Folution. Fig. 8A shows the kinetics of such a flash. Whereas the onset of light emission from the lumisomes appears to be sufficiently rapid to account for the in viva kinetics it is clear that the decay rate is consider- ably slower. This is also true when lumisomes are prelysed in distilled water followed by the addition of calcium ions (Fig. 8-8). It is interesting that the rates of both the onset and decay of luminescence is greatly decreased in the case of the :oluble photoprotein of Renilla (Fig. 8B).

Hastings et al. (25) made a study of the kill&c response of Aequorea photoprotein to rapid changes in calcium concentra- tion. They presented kinetic evidence which suggest that there is an initial equilibrium established between the soluble photo- protein of Aequorea and calcium ion and that the luminescence decay is dependent upon the continual presence of calcium ion. Removal of calcium ion during the luminescence reaction resulted

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SECONDS

1 FIG. 8. Kinetics of the bioluminescent reactions of Renilla reni- formis extracts. Lumisomes and soluble photoprotein were sus- pended in 0.1 ml of the homogenizing medium (see “Methods”). Point of initiation of reaction is denoted by an arrow. A, lumi- somes were lysed with a hypotonic calcium solution (0.4 ml of 0.01 M CaC12); B, soluble photoprotein was triggered with a hypo- tonic calcium solution (as in A); C, lumisomes were given 0.2 ml of an isotonic calcium solution (NaCl, 0.6 M; ca&, 0.05 M) fol- lowed by lysis with 0.4 ml of Hz0 (D) ; E, lumisomes were prelysed with 0.4 ml of H20 followed by calcium chloride (0.2 ml of 0.05 M

CaCb) injection; F, lumisomes were lysed with a hypotonic cal- cium solution (as in A) followed by injection of 0.1 ml of 0.1 M

EGTA, pH 7.8. The curve represents tracings of oscilloscope read- out from the Nova computer (19). The concentration of photo- protein was adjusted to give similar intensities at each treatment.

in a large increase in the decay rate. The experiment illustrated in Fig. 8F suggest that the same phenomenon may be occurring in the lumisomes. When non-isotonic calcium is added the typical rapid onset of luminescence is observed. When a sec- ondary molar excess addition of EGTA is made the initial lu- minescence decay is greatly increased and now appears more rapid than the in viva decay.

These experiments suggest that the photocytes exert careful control over the movement of calcium ions to and from the ap- propriate sites within the lumisomes. Such a control could esplain the kinetics of in z&o bioluminescent flashes. A similar conclusion has been drawn recently based on work with soluble photoproteins (15).

Distribution of Lumisomes-Lumisomes were found to occur in all the species of Anthozoa and Hydrozoa studied (Table III). As shown by the data in Table III lumisomes from the different species were essentially similar in their characteristics. For example they all produce light when in the presence of non- isotonic calcium, they all band on sucrose at a density of ap- proximately 1.17 g cm+ and the lumisomes from all of the species examined produced green light (X,,, = 509 nm) which is typical of the in viva emissions of these animals. Furthermore Renilla-

like luciferases could be extracted from all lumisomes derived from the Anthozoa but this was not the case for lumisomes iso- lated from the Hydrozoa. This observation is in agreement with recent data of Cormier et al. (16).

TAHLE III

Conpzrison of Iumisomes from Cnidaria

Particles isolated from

A. Class Hydrozoa Obelia longissima Obelia geniculata Clytia edwardsia

B. Class Anthozoa Subclass Alcyonaria Plilosarcus guernyi

Stylatula elongata Acanthoptilum gTCICile

Renilla reniformis Renilla mtilleri Renilla kiillikeri

Subclass Zoantharia Paraaoanthus ZuciJi-

cum

Activity ependent n calciurr

ion

+’ + +

+’

+ + +

+ +

+

ktivity lepend- ent on particle lysis”

+ + +

+ + + + + +

+

-

Band on sucrose

gradient

I-

? ?

+

+ + + + + +

?

0

1

PIeSenCe ‘f Renilla

type uciferase

(9)

-

-

-

+

+

+

+

+

+

+

Light emis- sion

greer?

? + ?

+ + + + + +

?

u Photoprotein activity which was not dependent on particle lysis was also present in all preparations but represented less than 10% of the particle luminescence.

b Color of bioluminescence was obtained as described in Fig. 1 except Obelia geniculata where total light passing through variable wedge interference filter (Oriel Optics Co.) was compared at 509 nm and 470 nm.

c f, characteristic was present; -, characteristic was absent; ?. insufficient material was obtained to make determination.

Comments on Physiological SignQicance of Lumisome-From the experiments described above it is clear that lumisomes con- tain all of the necessary proteins required to carry out the type of bioluminescence observed in vivo, i.e. photoprotein, luciferase, and a green fluorescent protein. Furthermore these proteins appear to be membrane-bound within the lumisome. This is an important consideration in producing the observed green emis- sion. Wampler et al. (26) have shown that the production of green light in vitro is highly dependent upon the concentration of luciferase and the green fluorescent protein. That is only at sufficiently high concentrations of each protein does one observe green emission. Previous calculations have shown that the critical transfer distance necessary to account for the efficiency of transfer seen is 28 A (10). To achieve this transfer distance would require in vitro protein concentrations t-70 to three orders of magnitude greater than those we are using. A logical ex- planation involves protein-protein interaction between the green fluorescent protein and luciferase or soluble photoprotein (26). The lumisome membrane provides an excellent environment for such interaction and complex formation to occur.

In the case of Renilla, luciferase may function as a precursor photoprotein. Renilla photoprotein is viewed as a stabilized luciferase-luciferin complex. The finding that oxygen is also required for the Renilla photoprotein reaction and that the colors of bioluminescence are the same for both the luciferase-luciferin and photoprotein reactions lends credence to this suggestion. Thus within the lumisome the calcium-triggered photoprotein complexed to the green fluorescent protein is viewed as the func- tional light emitting unit.

To account for the kinetics of the in vivo bioluminescent flash the photocytes must. exert careful control over the movement of calcium ion into and out of the lumisome. A possible mech-

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anism for such control involves active transport of calcium ion across the Iumisome membrane.

The widespread occurrence of lumisomes among the coelen- terates suggests that these subcellular part.icles or organelles represent the cellular site of bioluminescence in all coelenterates.

Acknowledgments-We wish to thank Dr. Kazuo Hori for supplying Renilla luciferin, Dr. J. E. Wampler for providing the absolute fluorescence and bioluminescence emission data, and Mr. B. Spurlock for the electron micrograph of Renilla lumisomes.

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James M. Anderson and Milton J. CormierLumisomes, the Cellular Site of Bioluminescence in Coelenterates

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