Proc. Nat. Acad. Sci. USA Vol. 72, No. 7, pp. 2530-2534, July 1975 Biochemistry Extraction of Renilla-type from the -activated , mnemiopsin, and berovin* (/sea pansy//peroxide/ctenophore) WILLIAM W. WARD AND MILTON J. CORMIER Bioluminescence Laboratory, Department of Biochemistry, University of Georgia, Athens, Ga. 30602 Communicated by W. D. McElroy, April 3,1975

ABSTRACT Photoproteins, which emit in an oxy- strictly dependent on the concentration of molecular oxy- gen-independent intramolecular reaction initiated by calci- gen. The bioluminescence reaction scheme for an anthozoan um ions, have been isolated from several bioluminescent or- coelenterate, the sea pansy Renilla reniformis, is an example ganisms, including the hydrozoan Aequorea and the ctenophore Mnemiopsis. The system of a related anthozoan of the luciferin-luciferase type system (4). coelenterate, the sea pansy Renilfa reniformis, however, is dependent, requiring two organic components, luci- Luciferin + 0. + luciferase (E) - ferin and luciferase. Previously published indirect evidence (490 nm) indicates that photoproteins may contain a Renilla-type 11ci- E-oxyluciferin + CO2 + hp ferin. reaction scheme of Aequorea as We have now extracted in high yield a Renilfa-type lucifer- We view the in from three photoproteins, aequorin (45% yield), mnem- follows (13): iopsin (98% yield), and berovin (85% yield). Photoprotein lu- ciferin, released from the holoprotein by mercaptoethanol Protein-chromophore + nCa++ treatment and separated from apo-photoprotein by gel filtra- + CO. + hi (469 nm) tion, no longer responds to calcium but now requires lucif- Ca,-protein-oxyluciferin erase and 02 for light production. Photoprotein luciferin is The apparent dissimilarities between luciferase-catalyzed identical to Renilla luciferin with respect to reaction kinetics reactions and photoprotein reactions have been perceived as and bioluminescence spectral distribution. have even In view of these results, the generally accepted hypothesis fundamental biochemical differences (1, 7) and that the photoprotein chromophore is a protein-stabilized hy- served as the basis for category assignment in several classifi- droperoxide of luciferin must be modified. We believe, in- cation schemes of bioluminescent systems (14-16). stead, that the chromophore is free luciferin and that oxygen Recent experiments indicate that photoproteins are not is bound as an oxygenated derivative of an amino-acid side biochemically unique, but are closely related to the luci- chain of the protein. We propose the general term "coelen- ferin-luciferase type systems of Cypridina and especially terate luciferin" to describe the light-producing chromophore Renilla (4, 13, 17). For example, Renilla luciferin in metha- from all bioluminescent coelenterates and ctenophores. nol absorbs light maximally at 435 nm, the peak of mnem- Photoproteins from several of bioluminescent coelen- iopsin absorption. In aprotic solvents Renilla luciferin ab- terates (1-4) and ctenophores (2, 4, 5) emit a rapid flash of sorbs at 454 nm, the absorption maximum of aequorin. The light in an oxygen-independent intramolecular reaction ini- Renilla luciferin reaction product and the products of Ca++- tiated by calcium ions (1, 6). Additional organic cofactors or induced light emission from aequorin and mnemiopsin all substrates are not required for light production, and photo- absorb in the near UV with a maximum at 335 nm (7, 8, 17). proteins have not been shown to turn over in vitro. The pho- The of a fully active analog of Renilla luciferin toproteins, aequorin, from the hydrozoan jellyfish Aequorea, (I, Fig. 1) has been elucidated and confirmed by total syn- and mnemiopsin, from the ctenophore Mnemiopsis, have thesis (17, 18). This analog is identical to native Renilla luci- been purified to homogeneity (5, 6). Berovin, from the cten- ferin except that the benzyl side chain at position 2 is re- ophore Beroe (5), has been obtained in 50% pure form. Each placed with a substituted phenolic side chain. For conve- is a single polypeptide chain having a molecular weight be- nience of discussion this analog will be referred to as Renilla tween 25,000 and 30,000. Aequorin has a visible absorption luciferin. The products of the light reaction, CO2 and oxylu- band with a peak near 460 nm (7), while mnemiopsin has a ciferin (II, Fig. 1), have also been determined (11, 17). With corresponding absorption band with a peak near 435 nm (8). the possible exception of the position 2 substituent men- In each case, the visible absorption band is lost during the tioned above, Renilla oxyluciferin is identical to Aequorea light-producing reaction with Ca++ and a product having an oxyluciferin (III, Fig. 1) (19). Furthermore, the hydrolysis absorption maximum at 335 nm is formed. products of both oxyluciferins, termed AF-350 (20) or Renil- Photoprotein reactions appear to be distinctly different la etioluciferin (IV, Fig. 1), are identical in all respects. In from the classical luciferin-luciferase reactions of the addition, it has been shown that a Renilla-type luciferyl sul- (Photinus), sea pansy (Renilla), and crustacean (Cypridina). fate (V, Fig. 1), the storage form of luciferin, is present in In these systems the light-producing reaction is the - tissue extracts of Aequorea and Mnemiopsis (21). catalyzed oxidative decarboxylation of a low-molecular- Preliminary communications (13, 22) reported the pres- weight luciferin molecule (4, 9-12). The light intensity is ence in pure samples of aequorin and mnemiopsin of luci- ferin molecules that crossreact with Renilla luciferase. In * This is no. XVI of a series entitled "Studies on the Biolumines- this paper we describe the results of improved methods for cence of Renilla reniformis." extracting photoprotein luciferin. The properties of luciferin 2530 Downloaded by guest on October 1, 2021 Biochemistry: Ward and Cormier Proc. Nat. Acad. Sci. USA 72 (1975) 2531

0 -CH0 _~OH*C-H lkC-CH2' 1 2. %N NH

JN

I I

N NH2 .3: N 'C2

CH2

FIG. 1. of synthetic Renilla luciferin (I), Renilla (II), and Aequorea (III) oxyluciferins and etioluciferins (IV), luciferyl sulfate (V), and luciferin hydroperoxide (IV).

isolated from aequorin, mnemiopsin, and berovin are de- To extract luciferin from aequorin, the photoprotein was scribed and implications concerning the role of oxygen in first treated at 220 by the method of Shimomura et al. (28), photoprotein reactions are discussed. with 2 mM NaHSO3 in 50 mM sodium phosphate buffer (pH 6.0) containing 5 mM EDTA. The 30-min bisulfite in- cubation, and all treatments, were MATERIALS AND METHODS subsequent performed anaerobically under argon. Although ethyl ether extracts of The photoproteins aequorin, mnemiopsin, and berovin were the bisulfite-treated aequorin were inactive with luciferase, purified as described (5, 24). Renilla luciferase was exten- later treatments with methanol, urea, or mercaptoethanol sively purified and was free of contamination from luciferin yielded active luciferin. Highest yields were produced by binding protein (23), luciferin sulfokinase (25), and green 3',-hr anaerobic treatment with 1.25 M 2-mercaptoethanol fluorescent protein (26, 27). Reagent grade chemicals were at 220. Luciferin was quantitatively released from the cteno- used throughout. phore photoproteins mnemiopsin and berovin by 30-min an-

Table 1. Extraction of Renilla-type luciferin from aequorin, mnemiopsin, and berovin

Calcium-dependent reaction Renilla luciferase-dependent reaction Total Total % Photo- photon mol of photon mol of protein chromo- yield* chromophore yield chromophore phore released as Experiment (Einsteins) reactedt (Einsteins) reacted* free luciferin A. Native aequorin 2.0 x 1012 6.9 X 10-12 <2 X 10-1 <3 X 10't 0.0 B. Aequorin + NaHSO3§ <2 x 10-18 <6 X 1018 2.5 X 10-'4 5.0 X 10-'3 7.3 C. B + 1.25 M 2-ME¶ <2 x 10-18 <6 x 10-18 1.5 X 10-s3 3.1 X 10-12 45 D. Native mnemiopsin 7.3 x 10-12 6.1 x 10-" 6.0 X 10'5 1.2 X 10-X3 0.2 E. D + 1.25 M 2-MEII <2 x 10-18 <1 X 10-'7 3.0 X 10-12 6.0 x 10-" 98 F. Native berovin 8.8 x 10-'4 7.4 x 10-13 <2 x 1018 <3 X 10-'7 0.0 G. F + 1.25 M 2-Mell <2 X 10-18 <1 X 10-17 3.2 x 10'4- 6.3 x 10-'3 85 The numbers tabulated represent the best yields obtained in a variety of experiments. Percentages in the last column were calculated for aequorin, mnemiopsin, and berovin as the ratio of mol of chromophore reacted in a luciferase-dependent reaction to the values 6.9 X 10-12, 6.1 X 10-11, and 7.4 X 10-13, respectively. No corrections have been made for luciferin losses due to autooxidation. * Aequorin assays were performed with 0.05 M calcium acetate, pH 6.0, at 0°. Mnemiopsin assays were performed with 0.10 M CaCl2, 0.20 M Tris.HCl, pH 8.5, at 00. The same buffer, adjusted to pH 8.0, was used for berovin assays. t Corrections were made for the 0.29 quantum yield of aequorin (35) and the 0.12 quantum yield of mnemiopsin (Ward and Seliger, in prep- aration). A quantum yield of0.12 was assumed for berovin. t Corrections were made for the 0.05 quantum yield of Renilla luciferin (17). § Sodium bisulfite incubations were performed as described (28), but under anaerobic conditions. WAfter 30-min bisulfite treatment, 0.1 volume of 2-mercaptoethanol (2-ME) was added, making the final concentration 1.25 M mercapto-. ethanol. Anaerobic incubation was continued for 3% hr at room temperature. 11 Anaerobic incubation was run at room temperature for 30 min. Downloaded by guest on October 1, 2021 .r 2532 Biochemistry: Ward andCormier Proc. Nat. Acad. Sci. USA 72 (1975) Table 2. Spectral comparisons of luciferase-catalyzed bioluminescence from photbprotein Normalized light intensities with various sources of luciferin*t Renilla Aequorin Mnemiopsin Berovin Wavelength (nm) Luciferin Luciferin Photoprotein Luciferin Photoprotein Luciferin Photoprotein 440 0.186 0.174 0.640 0.199 0.275 0.182 0.275 460 0.563 0.590 0.937 490 1.00 1.00 0.936 1.00 0.980 1.00 01)980 550 0.546 0.522 0.276 590 0.109 0.092 0.096 0.113 0.170 0.101 0.170 * Each value under the headings "luciferin" represents the average of 10 luciferase-catalyzed light intensity assays normalized to the Renilla emission maximum at 490 nm. tValues under the headings "photoprotein," taken from published spectral distribution curves for the photoproteins aequorin (36), mnemiopsin (8), and berovin (8), have been normalized to the respective photoprotein emission maxima at 469, 485, and 485 nm.

aerobic treatment with 1.25 M mercaptoethanol in 10 mM A comparison of the spectral distribution shows TrisIHCl, 1 mM EDTA (pH 8.0) at 22°. Quantitative release that the extracted photoproteiri luciferins produce light on of mnemiopsin luciferin has also been achieved with 1.25 M reaction with Renilla luciferase distinctly different from the dithiothreitol in place of 1.25 M mercaptoethanol. parent photoprotein (Table 2). At the selected wavelengths, Bioluminescence emission spectra were compared with photoprotein luciferins emit the same relative light intensi- the use of interference filters (Ditric Optics), a technique ties as Renilla luciferin, suggesting that the spectra are iden- chosen for its sensitivity. Intensity measurements were nor- tical. malized to a reference measurement at the Renilla spectral Chromatograiphic Separation. Photoprotein luciferin is maximum (490 nm interference filter). A combined correc- generally bound to the apo-photoprotein very tightly. Dur- tion factor for filter and phototube was calculated at each ing purification, for.example, mnemiopsin is passed repeat- wavelength by taking the ratios of experimental and pub- edly over ion exchange and gel filtration columns without lished (21) values for relative spectral intensity of Renilla loss of the bound chromophore. The properties of mercap- bioluminescence. toethanol-treated mnemiopsin, however, suggest that mer- Chromatographic separations were performed on a Bio- captoethanol treatment promotes the dissociation of lucifer- Gel P-6 gel filtration column equilibrated with 1.25 M mer- in from apo-photoprotein. To test this, mercaptoethanol- captoethanol in 10 mM Tris-HCl, 1 mM EDTA buffer at pH treated mnemiopsin was chromatographed at 220 on Bio- 8.0. The 1.0 cm X 40 cm column was operated at 30 ml Gel P-6 (molecular weight exclusion 6000). Most of the mne- hr-1. miopsin chromophore (89%) chromatographed as free luci- ferin, appearing after the salt volume. Only 6% eluted in the RESULTS AND DISCUSSION Extraction of Photoprotein Luciferin. Recently (28) Shi- 0 l-or momura et al. described a "new" chromophore isolated '0 from pure aequorin by. treatment of the photoprotein with .u NaHSO3. This ether-soluble material, called "yellow com- Ul 0.8+ W pound," resembles Renilla luciferin physically and spectral- z 0 ly. By the method of Shimomura et al. for extracting "yel- I.- 0 0.6k low compound," we obtained a luciferin from pure aequorin I 0. and mnemiopsin that reacts with Renilla luciferase (13). By I- suitable modifications we have now isolated luciferin in high 0.4A yield not only from aequorin, but also from mnemiopsin and berovin (Table 1). Yields of luciferin from aequorin, by any z 0.2 method, were variable and appear to depend upon the ini- z tial concentration of photoprotein. Yields from mnemiopsin I- 02 I L.ase (using 1.25 M mercaptoethanol) were consistently high, re- C3 nn I.'s \ t~.-L'ase gardless of starting photoprotein concentration. In 13 experi- -J o'1 1I 2I 3I 4 441 ments the average luciferin yield from mnemiopsin was 88%. -tIME (MINUTES) FIG. 2. Mnemiopsin luciferin: light intensity dependence on Properties of Photoprotein Luciferin. The extracted luciferase and 02 concentrations. The reaction was initiated (I) by photoprotein luciferins no longer flash upon the addition of addition of 2,g of pure Renilla luciferase (L'ase) to a deaerated Ca++. Light production instead requires Renilla luciferase, buffer solution containing 0.8 ug of mnemiopsin that had and the intensity depends upon molecular oxygen concen- been treated with 1.25 M mercaptoethanol. After 30 sec, oxygen tration. These relationships are depicted in Fig. 2 for mnem- (02) was bubbled into the sample, followed by argon (Ar) 2 min iopsin luciferin. The reaction rate of Renilla luciferase with later. Second and third additions of luciferase (5 ,ug each) were made later during the reaction. Calcium addition (2 mM Ca++) did photoprotein luciferin is comparable to the rate at which lu- not initiate a photoprotein reaction, but rather inhibited slightly ciferase reacts with Renilla luciferin under the same condi- the luciferase reaction. Calcium inhibition of luciferase at this con- tions. Both require about 2 min for 90% decay from the centration has previously been observed (John Matthews, personal peak intensity (at low luciferase concentration). communication). Downloaded by guest on October 1, 2021 Biochemistry: Ward and Cormier Proc. Nat. Acad. Sci. USA 72 (1975) 2533

2A HRP Na Cl > 2.0 0 1.6O z o 1.2 0 .

.8

C

20 2-ME - TREATED

20 2-~E TE ATVOUED ( l 012400 0 5 0 25MCK~~~~~EUN OUE(l

FI..i-Gl -6croatgrpy(-AE.8eaef -erapoehno mn osi.Teclbaonfclunvdvlmewt hosrdihprxiae(RP meipin2isn heclbato fsatvlmewt a~ r honi anlA hecrmtormo traedfr i wt 12 MmrapothnlatO, sshw n aelB PnlC sth hoxaoga o nmipin irtteae fr3 mina22'ith .25 Mmercptoetano)

void volume, indicating that this small fraction of luciferin cence, it has been suggested (17, 29, 32-34) that in photo- had remained bound to protein (Fig. 3C). proteins the luciferin exists as a protein-stabilized luciferin In a separate experiment (Fig. SB) mnemiopsin pretreated hydroperoxide. The protein is viewed as protecting the reac- for 5 min was chromatographed at 00 on Bio-Gel P-6 equili- tive intermediate and serving as a receptor for Ca++ ions. brated with buffer containing 1.25 M mercaptoethanol. The This attractive model would not only explain the oxygen in- void volume protein eluted in 25 min, making the total time dependency of photoproteins, but would also explain why of exposure to mercaptoethanol 30 min. In contrast, virtually previous attempts to release a fused ring chromophore from all of the mnemiopsin chromophore (86%) remained associ- aequorin have been unsuccessful. As our data show, this ated with protein in the void volume. However, of this, only model is no longer tenable, and an alternative model is re- a small portion (<5%) responded as native photoprotein by quired. flashing on the addition of Ca++. The remaining portion We have recently shown that Renilla luciferin can exist in had been altered by mercaptoethanol so that now it required several tautomeric forms (13), one with an absorption maxi- not only Ca++, but also luciferase and oxygen for biolumi- mum at 435 nm (tautomer A) and another at 454 nm (tau- nescence. In these respects the void volume protein resem- bles the Renilla luciferin binding protein (23). The calcium dependency of this altered mnemiopsin can be overcome by warming it to room temperature in the presence of mercap- CTENOPHORE PHOTOPROTEIfI 2- LUCIFERIN BINDING PROTEIN toethanol, with the quantitative release of bound luciferin. A I 00 schematic diagram of the release of luciferin from cteno- I CA++ OR HEAT phore photoproteins, incorporating the "binding protein" in- CA termediate, is pictured in Fig. 4. COELENTERATE LUCI FERIN' Oxygen Involvement. An oxygenated intermediate in the bioluminescent and chemiluminescent oxidations of Renilla I LUCIFERASE + 02 (11, 29) and Cypridina (12, 29-31) luciferins has been pro- posed as the linear hydroperoxide at position 2 in the imid- LIGHT ( =A,. 485 NM) LIGHT (XMAX - 490 NM) azole ring (VI, Fig. 1). In view of the striking similarities be- FIG. 4. Model for the release of luciferin from ctenophore pho- tween aequorin bioluminescence and Renilla biolumines- toprotein. 2-ME, 2-mercaptoethanol. Downloaded by guest on October 1, 2021 2534 Biochemistry: Ward and Cormier Proc. Nat. Acad. Sci. USA 72 (1975)

tomer B). In water and methanol the A form predominates, 4. Cormier, M. J., Hori, K. & Anderson, J. M. (1974) Biochim. but in aprotic solvents form B is the major species. The tau- Blophys. Acta 346,137-164. tomeric equilibrium is shown below: 5. Ward, W. W. & Seliger, H. H. (1974) Biochemistry 13, 1491-1499. 6. Kohama, Y., Shimomura, 0. & Johnson, F. H. (1971) Bio- A B chemistry 10, 4149-4152. 7. Shimomura, 0. & Johnson, F. H. (1969) Biochemistry 8, H 3991-3997. 0 8. Ward, W. W. & Sliger, H. H. (1974) Biochemistry 13, 1500- 1510. I R3 9. Plant, P. J., White, E. H. & McElroy, W. D. (1968) Blochem. N N Blophys. Res. Commun. 31,98-103. 10. DeLuca, M. & Dempsey, M. E. (1970) Biochem. Blophys. Res. No N;R Commun. 40,117-122. RI N R2 11. DeLuca, M., Dempsey, M. E., Hori, K., Wampler, J. E. & Cormier, M. J. (1971) Proc. Nat. Acad. Sd. USA 68, 1658- H 1660. 12. Stone, H. (1968) Biochem. Biophys. Res. Commun. 31,386- Presence of a peroxide group at position 2 of luciferin (VI, 391. Fig. 1) would fix the electronic structure into a tautomeric 13. Hori, K., Anderson, J. M., Ward, W. W. & Cormier, M. J. (1975) Biochemistry, in press. form nm resembling B, the 454 species. Therefore, the linear 14. Johnson, F. H. (1967) in Comprehensive Biochemistry, eds. hydroperoxide model for photoproteins would predict a visi- Florkin, M. & Stotz, E. H. (Elsevier Pub. Co., New York), Vol. ble absorption band centered near 454 nm (or red-shifted as 27, pp. 79-136. the result of increased electron delocalization contributed by 15. Hastings, J. W. (1968) Annu. Rev. Biochem. 37,597-630. the peroxide group). However, mnemiopsin absorbs maxi- 16. Johnson, F. H. & Shimomura, 0. (1972) in Photophysiology, mally at 435 nm, and in this respect resembles tautomer A. ed. Giese, A. C. (Academic Press, New York), Vol. 7, pp. Since tautomer A cannot accommodate a peroxide function 275-34. at position 2, the mnemiopsin chromophore is viewed as an 17. Hori, K., Wampler, J. E., Matthews, J. C. & Cormier, M. J. exception to the luciferin hydroperoxide model. (1973) Biochemistry 12, 443-4468. 18. Hori, K. & Cormier, M. J. (1973) in We recently proposed a new model and for photoproteins Bioluminescence, eds. Cormier, M. J., Hercules, D. M. & Lee, based primarily on the spectroscopic evidence cited above J. (Plenum Press, New York), pp. 361-369. (13). According to this model, a free Renilla-type luciferin is 19. Shimomura, 0. & Johnson, F. H. (1973) Tetrahedron Lett. 31, noncovalently bound to the protein. The luciferin assumes 2963-2966. the A configuration in mnemiopsin and the B form in ae- 20. Shimomura, 0. & Johnson, F. H. (1972) Biochemistry 11, quorin. To account for oxygen independency, an amino-acid 1602-1608. side chain, not the chromophore, is presumed to contain an 21. Cormier, M. J., Hori, K., Karkhanis, Y. D., Anderson, J. M., oxygenated species, possibly a hydroperoxide group. Calci- Wampler, J. E., Morin, J. G. & Hastings, J. W. (1973) J. Cell. um binding would then induce a conformational change, re- Physiol. 81, 291-298. sulting in transfer of this oxygenated species to the luciferin. 22. Ward, W. W., Hori, K. & Cormier, M. J. (1975) Biophys. J. (abstr.) 15, 53a. The quantitative release of Renilla-type luciferin from 23. Anderson, J. M., Charbonneau, H. & Cormier, M. J. (1974) three different photoproteins strengthens this proposal and Biochemistry 13, 1195-1200. greatly weakens the argument put forward by Shimomura 24. Brown, J. E. & Blinks, J. R. (1974) J. Gen. Physiol. 64, 643- et al. (28) for a separate "yellow compound," unrelated to 665. the light-emitting chromophore, but which serves as a hy- 25. Cormier, M. J., Hori, K. & Karkhanis, Y. D. (1970) Biochemis- drogen transfer acceptor. We suggest that photoprotein luci- try 9, 1184-1188. ferins are identical or very nearly identical to Renilla luci- 26. Wampler, J. E., Hori, K., Lee, J. W. & Cormier, M. J. (1971) ferin, and we propose the general term "coelenterate luci- Biochemistry 10, 2903-2910. ferin" to refer to the bioluminescent chromophore from all 27. Wampler, J. E., Karkhanis, Y. D., Hori, K. & Cormier, M. J. bioluminescent and (1972) Fed. Proc. 31,419. . 28. Shimomura, O., Johnson, F. H. & Morise, H. (1974) Biochem- istry 13,3278-3286. 29. McCapra, F. & Chang, Y. C. (1967) Chem. Commun., 1011- We gratefully acknowledge Dr. J. R. Blinks for his gift of pure 1012. aequorin, Mr. J. C. Matthews for preparing highly purified Renilla 30. Cormier, M. J., Wampler, J. E. & Hori, K. (1973) in Progress luciferase, and Dr. K. Hori for supplying Renilla luciferin. We also in the Chemistry of Organic Natural Products, eds. Herz, thank Dr. H. H. Seliger, who sponsored the original research on W., Grisebach, H. & Kirby, G. W. (Springer-Verlag, New ctenophore photoproteins under AEC Contract no. AT (11-U-3277). York), Vol. 30, pp. 1-60. This work was supported in part by the National Science Founda- 31. Shimomura, 0. & Johnson, F. H. (1971) Biochem. Biophys. tion and the U.S. Atomic Energy Commission. M.J.C. is a Career Res. Commun. 44,340346. Development Awardee, no. 1-K3-6M-3331-10 of the U.S. Public 32. Hastings, J. W. & Gibson, Q. H. (1963) j. Biol. Chem. 238, Health Service. Contribution no. 293 from the University of Geor- 2537-2554. gia Marine Institute, Sapelo Island, Ga. 33. McElroy, W. D. & Seliger, H. H. (1963) Adv. Enzymol. 25, 119-166. 34. Hastings, J. W. & Morin, J. G. (1969) Biochem. Biophys. Res. 1. Shimomura, O., Johnson, F. H.; & Saiga, Y. (1962) J. Cell. Commun. 37,493-498. Comp. Physiol. 59,223-240. 35. Shimomura, 0. & Johnson, F. H. (1970) Nature 227, 1356- 2. Morin, J. G. & Hastings, J. W. (1971) J. Cell. Physiol. 77, 1357. 305-312. 36. Morise, H., Shimomura, O., Johnson, F. H. & Winant, J. 3. Campbell, A. K. (1974) Biochem. J. 143,411-418. (1974) Biochemistry 13,2656-2662. 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