Properties of the bioluminescent protein aequorin

Shimomura, Osamu; Johnson, Frank H.

doi:10.1021/bi00838a015

Cited by 137 publications

(98 citation statements)

References 10 publications

(15 reference statements)

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“…about 2 % greater than the peak of the emission spectrum of aequorin. Shimomura & Johnson (1969) The final concentration inside the axon of diameter 800 #t would then be about 0-14/uM which is too small to buffer the internal calcium or to interfere with calcium movements appreciably. From the optical density of the injection solution and the extinction coefficient given by Shimomura & Johnson (1969) the concentration of aequorin was calculated as about 100^M , on the assumption that no other substance was present.…”

Section: Calibration Of Light Recording #Y8temmentioning

confidence: 99%

“…The aequorin used in these experiments was collected, extracted and partially purified according to the procedures described by Shimomura & Johnson (1969). Further purification was carried out at University College London, again according to the procedures of Shimomura & Johnson, except that the ohromotography was only carried out twice on DEAE-cellulose and once on Sephadex.…”

Section: Aequorin Preparationmentioning

confidence: 99%

See 1 more Smart Citation

Depolarization and calcium entry in squid giant axons

Baker¹,

Hodgkin²,

Ridgway³

1971

The Journal of Physiology

703

381

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SUMMARY1. Changes in ionized calcium in giant axons were followed by recording the light produced by injected aequorin.2. From the effect of injecting calcium buffers the internal concentration of ionized calcium was found to be about the same as in a mixture of 45 Ca EGTA: 55 free EGTA, i.e. about 04a1sM.3. After an axon had been exposedwto cyanide for 50-100 min the velocity of the aequorin reaction increased about 500 times. This effect, which could be reversed rapidly by removing cyanide, was probably brought about by release of calcium from an internal store.4. Injecting 30 tumole ATP per litre of axoplasm into a cyanide-poisoned axon caused a transient lowering of light intensity; oligomycin blocked the effect.5. Raising external cqicium or replacing external sodium by choline or lithium reversible increased the light produced by axons injected with aequorin.

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Section: Calibration Of Light Recording #Y8temmentioning

confidence: 99%

Section: Aequorin Preparationmentioning

confidence: 99%

Depolarization and calcium entry in squid giant axons

Baker¹,

Hodgkin²,

Ridgway³

1971

The Journal of Physiology

703

381

View full text Add to dashboard Cite

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“…As mentioned above, AF-350 (1) can be isolated when the calcium-triggered photoprotein of Aequorea is treated with urea and mercaptoethanol (9). It is most interesting to find that this structure is an integral part of the structure of Renilla luciferin.…”

Section: Discussionmentioning

confidence: 94%

Structure and Chemical Synthesis of a Biologically Active Form of Renilla (Sea Pansy) Luciferin

Hori

Cormier

1973

Proc. Natl. Acad. Sci. U.S.A.

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The structure of a biologically active form of Renilla (sea pansy) luciferin has been elucidated; this structure, confirmed by total chemical synthesis, is 3,7-dihydro-2-methyl-6-(p-hydroxyphenyl)-8-benzylimidazo [1,2-al pyrazin-3-one. In the natural compound the methyl group at the 2 position is replaced by an unknown, more complex group. For this reason the synthetic compound is 10% as active as the natural compound in producing light with Renilla luciferase. However, the spectral properties of the two compounds are identical. In addition the rates of the luminescent reaction with both compounds are-similar, and the color of the light produced is identical in each case.A compound isolated from the calcium-triggered photoprotein aequorin has been identified by Shimomura and Johnson [(1972) Biochemistry 11, 16021 to be 2-amino-3-benzyl-5-(,i-hydroxyphenyl)pyrazine. This compound forms an integral part of the structure of Renilla luciferin. This, and other evidence, suggests that the structure elucidated for Renilla luciferin is a more general one associated with the luciferins of most, if not all, bioluminescent coelenterates.From about 40,000 sea pansies (Renilla reniformis), obtained by dredging the ocean bottom at depths of 10-20 meters, one can obtain about 0.5 mg of pure Renilla luciferin. It is for this reason that elucidation of the structure of Renilla luciferin has been a difficult problem. We have accumulated a considerable amount of absorption data (U.V., visible, I.R.), as well as chemical and high-resolution mass spectroscopy data, over the last 10 years (1-3). These data, and the observation that several of the physical and chemical properties of Renilla luciferin were similar to those of Cypridina luciferin (1), led us to propose a partial tentative structure for Renilla luciferin that was similar to the structure of Cypridina luciferin (1). Due to the small amount of Renilla luciferin available and the ease with which luciferin is autooxidized good NMR data have never been available to us. Thus, several alternative structures were possible based on the mass spectroscopy data.A choice between the various possible structures might have been a more difficult problem if it were not for several observations made by us and by other investigators in the field of bioluminescence who were working on problems that seemed at first unrelated. The key observations and ideas that led to the proper choice of structure are outlined below.When the luminous tissues of bioluminescent coelenterates are extracted with EDTA-containing buffers, a protein can be isolated that exhibits a bluish luminescence upon the addition of calcium ions (4-7). Such proteins were termed "photoproteins." Recently, the existence of calcium-triggered photoproteins have been demonstrated in extracts of a wide variety of coelenterates such as Obelia, Aequorea, Pelagia, Renilla, Mnemiopsis, Campanularia, Clytia, Phialidium, Lovenella, Ptilosarcus, and Diphyes (7). Furthermore, the characteristics of these photoproteins were for t...

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“…Therefore, In I =-k(A)nt + c. [7] in which Io is the light intensity without drugs. In the final form, we have…”

Section: Methodsmentioning

confidence: 99%

“…Purification of the protein and elucidation of the mechanism of light emission have been carried out by Johnson, Shimomura, and their colleagues (7). The purified protein is estimated to have a luminescence activity of 4.5 X 10'5 photons per mg of purified protein (dry weight) at 250.…”

mentioning

confidence: 99%

Reversible and irreversible inhibition by anesthetics of the calcium-induced luminescence of aequorin.

Kamaya¹,

Ueda²,

Eyring³

1977

Proc. Natl. Acad. Sci. U.S.A.

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The actions of various kinds of so-called membrane-stabilizing drugs on Ca2+-induced flash intensity of purified aequorin, a photoprotein obtained from Aequorea aequorea, were examined. All drugs used in this experiment, including inhalational anesthetics, tetracaine, chlorpromazine, and morphine, depressed flash intensity, and the inhibition increased with time. The inhibition followed a mixture of reversible and irreversible kinetics. A method of analyzing irreversible kinetics is presented. The dissociation constants for reversible inhibition and the rate constants for irreversible inhibition are presented for each agent.It is well known that the excitability of cell membranes is closely related to the presence of Ca2+ outside the membrane. The so-called membrane-stabilizing actions of local anesthetics, general anesthetics, tranquilizers, and narcotics are frequently explained by their antagonistic action on Ca2+ (1-6).The photoprotein aequorin extracted from jellyfish (Aequorea aequorea) emits light when free Ca2+ is present. Purification of the protein and elucidation of the mechanism of light emission have been carried out by Johnson, Shimomura, and their colleagues (7). The purified protein is estimated to have a luminescence activity of 4.5 X 10'5 photons per mg of purified protein (dry weight) at 250.The present study reports the inhibitory action of local and general anesthetics on the Ca2+-induced luminescence of purified aequorin. METHODSThe highly purified aequorin used in this study was Tetracaine HCl, chlorpromazine HCl, and morphine sulfate were separately dissolved in the 50 mM imidazole buffer, pH 6.3., and mixed with the aequorin solution at a constant temperature (300) in a water bath. Halothane and methoxyflurane were vaporized in nitrogen by a copper kettle in an anesthesia machine. The anesthetic concentrations were estimated by the ratio of the saturated anesthetic gas flow to the flow of the nitrogen diluent.'The concentrations were confirmed by gas chromatography. The anesthetic vapor was equilibrated with the aequorin solutions by slow bubbling of the gas through the solution.Aliquots (0.5 ml) were periodically taken from each anesthetic/aequorin solution and injected (with a microsyringe) into 1.0 ml of calcium solution in a plastic cuvette. The light intensity was measured with a photomultiplier (Hitachi-Perkin-Elmer) and recorded on a strip chart recorder. RESULTSAt pH 6.0, 0.5 mM Ca2+ produced maximal light intensity; 50 and 10 ,uM Ca2+ produced 50 and 5% of the maximal light intensity, respectively (Fig. 1). Samples (0.5

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Properties of the bioluminescent protein aequorin

Cited by 137 publications

References 10 publications

Depolarization and calcium entry in squid giant axons

Depolarization and calcium entry in squid giant axons

Structure and Chemical Synthesis of a Biologically Active Form of Renilla (Sea Pansy) Luciferin

Reversible and irreversible inhibition by anesthetics of the calcium-induced luminescence of aequorin.

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