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Nowadays the recombinant Ca2+ -regulated photoproteins originating from marine luminous organisms are widely applied to monitor calcium transients in living cells due to their ability to emit light on Ca2+ binding. Here we report the specific activities of the recombinant Ca2+ -regulated photoproteins-aequorin from Aequorea victoria, obelins from Obelia longissima and Obelia geniculata, clytin from Clytia gregaria and mitrocomin from Mitrocoma cellularia. We demonstrate that along with bioluminescence spectra, kinetics of light signals and sensitivities to calcium, these photoproteins also differ in specific activities and consequently in quantum yields of bioluminescent reactions. The highest specific activities were found for obelins and mitrocomin, whereas those of aequorin and clytin were shown to be lower. To determine the factors influencing the variations in specific activities the fluorescence quantum yields for Ca2+ -discharged photoproteins were measured and found to be quite different varying in the range of 0.16-0.36. We propose that distinctions in specific activities may result from different efficiencies of singlet excited state generation and different fluorescence quantum yields of coelenteramide bound within substrate-binding cavity. This in turn may be conditioned by variations in the amino acid environment of the substrate-binding cavities and hydrogen bond distances between key residues and atoms of 2-hydroperoxycoelenterazine.

Bioluminescent solid-phase analysis was proposed to monitor the selection process and to determine binding characteristics of the aptamer-target complexes during design and development of the specific aptamers. The assay involves Ca2+ -regulated photoprotein obelin as a simple, sensitive and fast reporter. Applicability and the prospects of the approach were exemplified by identification of DNA aptamers to cardiac troponin I, a highly specific early biomarker for acute myocardial infarction. Two structurally different aptamers specific to various epitopes of troponin I were obtained and then tested in a model bioluminescent assay.

Site-directed mutagenesis is a powerful tool to investigate the structure-function relationship of proteins and a function of certain amino acid residues in catalytic conversion of substrates during enzymatic reactions. Hence, it is not surprising that this approach was repeatedly applied to elucidate the role of certain amino acid residues in various aspects of photoprotein bioluminescence, mostly for aequorin and obelin, and to design mutant photoproteins with altered properties (modified calcium affinity, faster or slower bioluminescence kinetics, different emission color) which would either allow the development of novel bioluminescent assays or improvement of characteristics of the already existing ones. This information, however, is scattered over different articles. In this review, we systematize the findings that were made using site-directed mutagenesis studies regarding the impact of various amino acid residues on bioluminescence of hydromedusan Ca2+ -regulated photoproteins. All key residues that have been identified are pinpointed, and their influence on different aspects of photoprotein functioning such as active photoprotein complex formation, bioluminescence reaction, calcium response and light emitter formation is discussed.

Color variants of Ca2+ -regulated photoprotein obelin were shown to be an important tool for dual-analyte binding assay. To provide site-directed conjugation with biospecific molecules, several obelin color mutants carrying unique cysteine residues were obtained and characterized for their novel properties. A pair of obelins Y138F,A5C and W92F,H22E,D12C was found to be most suitable (in terms of high bioluminescent activity and stability) as reporters in simultaneous assay of two targets in a sample. Availability of SH-groups, accessible for chemical modification, essentially simplifies the synthesis of biospecific conjugates, increases their yield and conserves obelins' bioluminescence activity. Conjugates with immunoglobulin and oligonucleotide were produced and successfully applied in single nucleotide polymorphism genotyping.

The molecular mechanisms of the bioluminescence systems of the firefly, bacteria and those utilizing imidazopyrazinone luciferins such as coelenterazine are gradually being uncovered using modern biophysical methods such as dynamic (ns-ps) fluorescence spectroscopy, NMR, X-ray crystallography and computational chemistry. The chemical structures of all reactants are well defined, and the spatial structures of the luciferases are providing important insight into interactions within the active cavity. It is generally accepted that the firefly and coelenterazine systems, although proceeding by different chemistries, both generate a dioxetanone high-energy species that undergoes decarboxylation to form directly the product in its S1 state, the bioluminescence emitter. More work is still needed to establish the structure of the products completely. In spite of the bacterial system receiving the most research attention, the chemical pathway for excitation remains mysterious except that it is clearly not by a decarboxylation. Both the coelenterazine and bacterial systems have in common of being able to employ "antenna proteins," lumazine protein and the green-fluorescent protein, for tuning the color of the bioluminescence. Spatial structure information has been most valuable in informing the mechanism of the Ca2+ -regulated photoproteins and the antenna protein interactions.

Luminous organisms use different protein-mediated strategies to modulate light emission color. Here, we report the transient-state kinetic studies of the interaction between photoprotein clytin from Clytia gregaria and its antenna protein, cgreGFP. We propose that cgreGFP forms a transient complex with Ca(2+)-bound clytin before the excited singlet state of the coelenteramide product is formed. From the spectral distribution and donor-acceptor separation distance, we infer that clytin reaction intermediates may interact only with the middle side part of cgreGFP.

Herein, the synthesis and characterization of an alkyne-modified luciferin is reported. This bioluminescent probe was accessed using C-H activation methodology and was found to be stable in solution and capable of light production with firefly luciferase. The luciferin analogue was also cell permeant and emitted more redshifted light than d-luciferin, the native luciferase substrate. Based on these features, the alkynyl luciferin will be useful for a variety of imaging applications.

Luciferin regenerating enzyme (LRE) contributes to in vitro recycling of d-luciferin to produce persistent and longer light emission by luciferase. Luciferin binding domains I and II among LREs regarded as potential candidates for luciferin-binding sites. In this study, for the first time, amino acids T69, G75 and K77 located at luciferin binding domain I of LRE from L. turkestanicus (T-LRE) substituted by using site-directed mutagenesis. Single mutant T(69)R increased luciferase light output more than two-fold over a longer time in comparison with a wild-type and other mutants of T-LRE. Nevertheless, double mutant (K(77)E/T(69)R) increased the amount of bioluminescent signal more than two-fold over a short time. In addition, G(75)E, K(77)E and G(75)E/T(69) R mutants did not improve luciferin-luciferase in vitro bioluminescence. Based on our results, addition of K(77)E/G(75)E and K(77)E/G(75)E/T(69)R mutants caused intermediate changes in bioluminescence from in vitro luciferin-luciferase reaction. These findings indicated that the amino acids in question are possible to be located within T-LRE active site. It may also be suggested that substituted Arg69 (Arg218) plays an important role in luciferin binding and the existence of Gly75 as well as Lys77 is essential for T-LRE which has already evolved to have different functions in nature.

Ca(2+) -regulated photoproteins are responsible for the bioluminescence of a variety of marine coelenterates. All hydromedusan photoproteins are a single-chain polypeptide to which 2-hydroperoxycoelenterazine is tightly but non-covalently bound. Bioluminescence results from oxidative decarboxylation of 2-hydroperoxycoelenterazine, generating protein-bound coelenteramide in an excited state. The bioluminescence spectral maxima of recombinant photoproteins vary in the range 462-495 nm, despite a high degree of identity of amino acid sequences and spatial structures of these photoproteins. Based on studies of obelin and aequorin mutants with substitution of Phe to Tyr and Tyr to Phe, respectively [Stepanyuk GA et al. (2005) FEBS Lett 579, 1008-1014], it was suggested that the spectral differences may be accounted for by an additional hydrogen bond between the hydroxyl group of a Tyr residue and an oxygen atom of the 6-(p-hydroxyphenyl) substituent of coelenterazine. Here, we report the crystal structures of two conformation states of the F88Y obelin mutant that has bioluminescence and product fluorescence spectra resembling those of aequorin. Comparison of spatial structures of the F88Y obelin conformation states with those of wild-type obelin clearly shows that substitution of Phe to Tyr does not affect the overall structures of either F88Y obelin or its product following Ca(2+) discharge, compared to the conformation states of wild-type obelin. The hydrogen bond network in F88Y obelin being due to the Tyr substitution clearly supports the suggestion that different hydrogen bond patterns near the oxygen of the 6-(p-hydroxyphenyl) substituent are the basis for spectral modifications between hydromedusan photoproteins.

A novel luciferin from a bioluminescent Siberian earthworm Fridericia heliota was recently described. In this study, the Fridericia oxyluciferin was isolated and its structure elucidated. The results provide insight into a novel bioluminescence mechanism in nature. Oxidative decarboxylation of a lysine fragment of the luciferin supplies energy for light generation, while a fluorescent CompX moiety remains intact and serves as the light emitter.

Advances in bioanalytical techniques have become crucial for both basic research and medical practice. One example, bioluminescence imaging (BLI), is based on the application of natural reactants with light-emitting capabilities (photoproteins and luciferases) isolated from a widespread group of organisms. The main challenges in cardiac regeneration remain unresolved, but a vast number of studies have harnessed BLI with the discovery of aequorin and green fluorescent proteins. First described in the luminous hydromedusan Aequorea victoria in the early 1960s, bioluminescent proteins have greatly contributed to the design and initiation of ongoing cell-based clinical trials on cardiovascular diseases. In conjunction with advances in reporter gene technology, BLI provides valuable information about the location and functional status of regenerative cells implanted into numerous animal models of disease. The purpose of this review was to present the great potential of BLI, among other existing imaging modalities, to refine effectiveness and underlying mechanisms of cardiac cell therapy. We recount the first discovery of natural primary compounds with light-emitting capabilities, and follow their applications to bioanalysis. We also illustrate insights and perspectives on BLI to illuminate current efforts in cardiac regeneration, where the future is bright.

Proteorhodopsin (PR), a ubiquitous membrane photoprotein in marine environments, acts as a light-driven proton pump and can provide energy for bacterial cellular metabolism. However, knowledge of f ...

In the last few decades, bioluminescent systems based on the expression of a luciferase and the addition of a luciferin to monitor the emission of light have become very important tools for biological investigations. A growing proportion of these systems use coelenterazine or analogues of imidazo[1,2-a]pyrazine luciferins along with photoproteins or luciferases from sea creatures such as Aequorea, Renilla, Gaussia or Oplophorus. Central to the success of these tools are the synthetic pathways developed not only to prepare the naturally occurring luciferins, but also to design altered compounds that exhibit improved bioluminescence. Current work is indeed focused on the design of systems exhibiting extended luminescence ("glow" systems) or redshifted wavelengths, as well as constructions better adapted to conditions in cells or in vivo. This review describes the synthetic pathways used to prepare imidazo[1,2-a]pyrazine luciferins along with the research efforts aimed at preparing analogues even better suited to the design of assays.

Bioluminescence, the emission of light from live organisms, occurs in 18 phyla and is the major communication system in the deep sea. It has appeared independently many times during evolution but its origins remain unknown. Coelenterazine bioluminescence discovered in luminous jellyfish is the most common chemistry causing bioluminescence in the sea, occurring in seven phyla. Sequence similarities between coelenterazine luciferases and photoproteins from different phyla are poor (often < 5%). The aim of this study was to examine albumin that binds organic substances as a coelenterazine luciferase to test the hypothesis that the evolutionary origin of a bioluminescent protein was the result of the formation of a solvent cage containing just a few key amino acids. The results show for the first time that bovine and human albumin catalysed coelenterazine chemiluminescence consistent with a mono-oxygenase, whereas gelatin and haemoglobin, an oxygen carrier, had very weak activity. Insulin also catalysed coelenterazine chemiluminescence and was increased by Zn(2+). Albumin chemiluminescence was heat denaturable, exhibited saturable substrate characteristics and was inhibited by cations that bound these proteins and by drugs that bind to human albumin drug site I. Molecular modelling confirmed the coelenterazine binding site and identified four basic amino acids: lys195, arg222, his242 and arg257, potentially important in binding and catalysis similar to naturally occurring coelenterazine bioluminescent proteins. These results support the 'solvent cage' hypothesis for the evolutionary origin of enzymatic coelenterazine bioluminescent proteins. They also have important consequences in diseases such as diabetes, gut disorders and food intolerance where a mono-oxygenase could affect cell surface proteins.

Light-sensitive Ca2+-regulated photoproteins are responsible for the bright bioluminescence of ctenophores. Using functional screening, four full-size cDNA genes encoding the same 208-amino-acid polypeptide were isolated from two independent cDNA libraries prepared from two Beroe abyssicola specimens. Sequence analysis revealed three canonical EF-hand calcium-binding sites characteristic of Ca2+-regulated photoproteins, but a very low degree of sequence identity (27–29%) with aequorin-type photoproteins, despite functional similarities. Recombinant berovin was expressed in Escherichia coli cells, purified, converted to active photoprotein and characterized. Active berovin has absorption maxima at 280 and 437 nm. The Ca2+-discharged protein loses visible absorption, but exhibits a new absorption maximum at 335 nm. The berovin bioluminescence is blue (λmax = 491 nm) and a change in pH over the range 6.0–9.5 has no significant effect on the light emission spectrum. By contrast, the fluorescence of Ca2+-discharged protein (λex = 350 nm) is pH sensitive: at neutral pH the maximum is at 420 nm and at alkaline pH there are two maxima at 410 and 485 nm. Like native ctenophore photoproteins, recombinant berovin is also inactivated by light. The Ca2+ concentration–effect curve is a sigmoid with a slope on a log–log plot of ∼ 2.5. Although this curve for berovin is very similar to those obtained for obelin and aequorin, there are evident distinctions: berovin responds to calcium changes at lower concentrations than jellyfish photoproteins and its Ca2+-independent luminescence is low. Recombinant berovin was successfully expressed in mammalian cells, thereby demonstrating potential for monitoring intracellular calcium. Database The nucleotide sequences have been deposited in the GenBankTM/EBI Data Bank with accession numbers: apoberovin cDNA genes, JN673813 (BA1), JN673814 (BA2), JN673815 (BA3), JN673816 (BA4); fragment 18S rRNA, JN673817 (BA-rRNA5).

The syllid polychaete Odontosyllis phosphorea produces brilliant displays of green bioluminescence during mating swarms. We studied freshly collected individuals of O. phosphorea in the laboratory to understand the characteristics of its luminescent system. Light emission appeared as an intense glow after stimulation with potassium chloride, and was associated with secreted mucus. The mucus was viscous, blue in color, and exhibited a long-lasting glow that was greatly intensified by addition of peroxidase or ammonium persulfate. The emission spectrum of mucus-associated bioluminescence was unimodal, with a maximum emission in the green spectrum between 494 and 504 nm. The fluorescence emission spectrum was similar, but the fluorescence intensity was low unless it originated from mucus that had already produced light, suggesting that the oxidized product of the light production is the source of fluorescence. Individuals as small as 0.5–1.0 mm produced bioluminescence that was mainly internal and not secreted as mucus. The early occurrence of bioluminescence in the life cycle of members of O. phosphorea suggests that bioluminescence may be used for purposes other than attracting mates. The luminous system was functional at temperatures as low as −20°C and was degraded above 40°C. Mixing hot and cold extracts of the mucus did not result in reconstituting original levels of light emission. Additionally, mucus samples exposed to oxygen depletion by bubbling with argon or nitrogen were still able to produce intense bioluminescence. These results suggest that bioluminescence from the mucus may involve a photoprotein rather than a luciferin–luciferase reaction.

Amino acid analyses after acid hydrolysis have demonstrated that luciferin, the substrate of the bioluminescent reaction of the dinoflagellate Pyrocystis lunula and its precursor. P630, possess the same peptide moiety. The molecular weight of the resulting chromopeptides would be 5400.

Summary Luciferase is a general term for enzymes catalyzing visible light emission by living organisms (bioluminescence). The studies carried out with Photinus pyralis (firefly) luciferase allowed the discovery of the reaction leading to light production. It can be regarded as a two-step process: the first corresponds to the reaction of luciferase’s substrate, luciferin (LH2), with ATPMg 21 generating inorganic pyrophosphate and an intermediate luciferyl-adenylate (LH2-AMP); the second is the oxidation and decarboxylation of LH2-AMP to oxyluciferin, the light emitter, producing CO2, AMP, and photons of yellow-green light (550– 570 nm). In a dark reaction LH2-AMP is oxidized to dehydroluciferyl-adenylate (L-AMP). Luciferase also shows acyl-coenzyme A synthetase activity, which leads to the formation of dehydroluciferyl-coenzyme A (L-CoA), luciferyl-coenzyme A (LH2CoA), and fatty acyl-CoAs. Moreover luciferase catalyzes the synthesis of dinucleoside polyphosphates from nucleosides with at least a 3 0 -phosphate chain plus an intact terminal pyrophosphate moiety. The LH2 stereospecificity is a particular feature of the bioluminescent reaction where each isomer, D-LH2 or L-LH2, has a specific function. Practical applications of the luciferase system, either in its native form or with engineered proteins, encloses the analytical assay of metabolites like ATP and molecular biology studies with luc as a reporter gene, including the most recent and increasing field of bioimaging. 2008 IUBMB IUBMB Life, 61(1): 6–17, 2009

Renilla luciferase (RLUC) is a versatile tool for gene expression assays and in vivo biosensor applications, but its catalytic mechanism remains to be elucidated. RLUC is evolutionarily related to the alpha/beta hydrolase family. Its closest known homologs are bacterial dehalogenases, raising the question of how a protein with a hydrolase fold can function as a decarboxylating oxygenase. Molecular docking simulations with the coelenterazine substrate against an RLUC homology model as well as a recently determined RLUC crystal structure were used to build hypotheses to identify functionally important residues, which were subsequently tested by site-directed mutagenesis, heterologous expression, and bioluminescence emission spectroscopy. The data highlighted two triads of residues that are critical for catalysis. The putative catalytic triad residues D120, E144, and H285 bear only limited resemblance to those found in the active site of aequorin, a coelenterazine-utilizing photoprotein, suggesting that the reaction scheme employed by RLUC differs substantially from the one established for aequorin. The role of H285 in catalysis was further supported by inhibition using diethylpyrocarbonate. Multiple substitutions of N53, W121, and P220--three other residues implicated in product binding in the homologous dehalogenase Sphingomonas LinB--also supported their involvement in catalysis. Together with luminescence spectra, our data lead us to propose that the conserved catalytic triad of RLUC is directly involved in the decarboxylation reaction of coelenterazine to produce bioluminescence, while the other active-site residues are used for binding of the substrate.

Phrixotrix (railroad worm) luciferases produce bioluminescence in the green and red regions of the spectrum, depending on the location of the lanterns, and are the only luciferases naturally producing red bioluminescence. Comparison of the luciferase sequences showed a set of substitutions that could be involved in bioluminescence colour determination: (a) unique substitutions in the red luciferase replacing otherwise invariant residues; (b) conserved basic residues in the green–yellow emitting luciferases; and (c) an additional R353 residue in red-emitting luciferase (Viviani et al., 1999). To investigate whether these sites have a functional role in bioluminescence colour determination, we performed a site-directed mutagenesis. Natural substitutions in the region 220–344 and residues in the putative luciferin-binding site were also investigated. With the exception of the previously identified substitution of R215 and T226 (Viviani et al., 2002), which display dramatic red-shift effects on the spectrum of green–yellow-emitting luciferases, only a few substitutions had a moderate effect on the spectrum of the green-emitting luciferase. In contrast, no single substitution affected the spectrum of the red-emitting luciferase. The results suggest that the identity of the active site residues is not so critical for determining red bioluminescence in PxRE luciferase. Rather, the conformation assumed during the emitting step could be critical to set up proper interactions with excited oxyluciferin. Copyright © 2007 John Wiley & Sons, Ltd.

Blue fluorescent protein from the calcium-binding photoprotein aequorin (BFP-aq) is a complex of Ca2+-bound apoaequorin and coelenteramide, and shows luminescence activity like a luciferase, catalyzing the oxidation of coelenterazine with molecular oxygen. To understand the catalytic properties of BFP-aq, various fluorescent proteins (FP-aq) have been prepared from semi-synthetic aequorin and characterized in comparison with BFP-aq. FP-aq has luciferase activity and could be regenerated into native aequorin by incubation with coelenterazine. The results from substrate specificity studies of FP-aq using various coelenterazine analogues have suggested that the oxidation of coelenterazine by BFP-aq in the luciferase reaction and the regeneration process to aequorin might involve the same catalytic site of BFP-aq.

An overview of the rhodopsin crystal structure provides a structural basis for understanding the structures and functions of other G-protein coupled receptors (GPCRs). All of the structural details observed to date for rhodopsin will not necessarily carry over to other GPCRs, but major features such as the arrangement of the seven transmembrane helices, the retinal/ligand binding site, the D(E)RY and NPXXY sequence and structural motifs, and the bent helices are likely characteristics of the GPCRs most closely related to rhodopsin. A general view of these structural features is presented here.

The crystal structures of calcium-loaded apoaequorin and apo-obelin have been determined at resolutions 1.7 A and 2.2 A, respectively. A calcium ion is observed in each of the three EF-hand loops that have the canonical calcium-binding sequence, and each is coordinated in the characteristic pentagonal bipyramidal configuration. The calcium-loaded apo-proteins retain the same compact scaffold and overall fold as the unreacted photoproteins containing the bound substrate, 2-hydroperoxycoelenterazine, and also the same as the Ca2+-discharged obelin bound with the product, coelenteramide. Nevertheless, there are easily discerned shifts in both helix and loop regions, and the shifts are not the same between the two proteins. It is suggested that these subtle shifts are the basis of the ability of these photoproteins to sense Ca2+ concentration transients and to produce their bioluminescence response on the millisecond timescale. A mechanism of intrastructural transmission of the calcium signal is proposed.

The photoprotein aequorin emits light by an intramolecular reaction in the presence of a trace amount of Ca(2+). Semi-synthetic aequorins, produced by replacing the coelenterazine moiety in aequorin with the analogues of coelenterazine, show widely different sensitivities to Ca(2+). To understand the structural basis of the Ca(2+)-sensitivity, we determined the crystal structures of four semi-synthetic aequorins (cp-, i-, br- and n-aequorins) at resolutions of 1.6-1.8 A. In general, the protein structures of these semi-synthetic aequorins are almost identical to native aequorin. Of the four EF-hand domains in the molecule, EF-hand II does not bind Ca(2+), and the loop of EF-hand IV is clearly deformed. It is most likely that the binding of Ca(2+) with EF-hands I and III triggers luminescence. Although little difference was found in the overall structures of aequorins investigated, some significant differences were found in the interactions between the substituents of coelenterazine moiety and the amino acid residues in the binding pocket. The coelenterazine moieties in i-, br-, and n-aequorins have bulky 2-substitutions, which can interfere with the conformational changes of protein structure that follow the binding of Ca(2+) to aequorin. In cp-aequorin, the cyclopentylmethyl group that substitutes for the original 8-benzyl group does not interact hydrophobically with the protein part, giving the coelenterazine moiety more conformational freedom to promote the light-emitting reaction. The differences of various semi-synthetic aequorins in Ca(2+)-sensitivity and reaction rate are explained by the capability of the involved groups and structures to undergo conformational changes in response to the Ca(2+)-binding.

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin (BFP-aq) was prepared and determined to be a heat resistant enzyme, catalyzing the luminescent oxidation of coelenterazine (luciferin) with molecular oxygen as a general luciferase. After treatment with excess ethylenediaminetetraacetic acid to remove Ca2+ from BFP-aq, the blue fluorescence shifted to a greenish fluorescence. This greenish fluorescent protein (gFP-aq) was identified as a non-covalent complex of apoaequorin with coelenteramide (oxyluciferin) in a molar ratio of 1:1. By incubation with coelenterazine in the absence of reducing reagents, gFP-aq was converted to aequorin at 25 °C. BFP-aq and gFP-aq possessing both fluorescence and luminescence activities may work as novel reporter proteins.

The Ca 2+ -regulated photoprotein obelin was substituted at Trp92 by His, Lys, Glu, and Arg. All mutants fold into stable conformations and produce bimodal bioluminescence spectra with enhanced contribution from a violet emission. The W92R mutant has an almost monomodal bioluminescence ( λ max =390 nm) and monomodal fluorescence ( λ max =425 nm) of the product. Results are interpreted by an excited state proton transfer mechanism involving the substituent side group and His22 in the binding cavity.

The presence of luminous organs in a few insects, belong-ing to different families and orders, offers a parallel case ofdifficulty (to the origin of the electric organs of fishes).Thus wrote Charles Darwin in ‘Difficulties on Theory’ in On theOrigin of Species (1). He couldn’t see how small change by smallchange could lead, apparently out of the blue, to a completelynew phenomenon such as the electric organs of fishes or theluminous organs of fireflies and jellyfish through his BIG idea ofnatural selection. A particular puzzle, pointed out by Darwin, isthe apparently random distribution of luminous species within aphylum, or even a genus. This problem was highlighted by E.N.Harvey, who likened the phylogenetic distribution of biolumines-cence to ‘a handful of wet sand being thrown at a blackboardcontaining a list of all genera’ (2), the luminous organisms beingidentifiedbywherethesandstuck.Thedistinguished astrophysistFred Hoyle argued strongly that Darwin’sideawouldnotwork.The mathematics just didn’t add up. Hoyle compared evolutionthrough random mutations to how ‘a tornado sweeping througha junk-yard might assemble a Boeing 747 from the materialstherein’ (3). But, the recent article published in the May –June issueof Luminescence (4) shines light on both Darwin’sandHoyle’sdilemma. Far from being a problem for evolution, this latest papershowsthatbioluminescenceisamodelforoneofthekeyproblemsin evolution – the origin of a new enzyme. All that is needed is asolvent cage, within which are just a few critical amino acids (4).Bioluminescence, or ‘phosphorescence’ as Darwin called it,is the emission of visible light from living organisms (5). Itoccurs in 8 phyla, Protozoa, Cnidaria, Ctenophora, Mollusca,Echinodermata, Arthropoda, Chaetognatha and Chordata,and is a major communication system in the deep sea. Allbioluminescence is chemiluminescence, the generic reactioninvolving the oxidation of a small organic molecule, the luciferin,catalysed by an enzyme, the luciferase, or photoprotein in somecases. All bioluminescence requires oxygen, in some form,because the energy required to excite an electron for theemission of visible light comes from oxidation. This is true evenin fireflies and glow-worms, which use ATP to form the realluciferin, AMP–luciferin. ATP does not have an energy-rich bond,and is not the energy source for firefly or any other biolumines-cence, as is often wrongly stated in several textbooks andsome websites! There are five recognized chemical families ofluciferin causing bioluminescence: flavines (e.g. bacteria);imidazopyrazines (e.g. hydroids, jellyfish, sea pansies and pens,decapod shrimp, some squid and fish);benzothiazole (e.g.firefliesand glow-worms); linear tetrapryolles (e.g. dinoflagellates andeuphausid shrimp); and aldehydes (e.g. earthworms and the snailLatia).However,theremustbeseveralotherchemistriesthathaveyet to be revealed, as these uncharacterized bioluminescent sys-tems do not cross-react with known luciferases. This molecularbiodiversitymeansthateachtypeofchemistrymusthaveevolvedindependentlyfroma differentorigin.Yettheevolutionaryoriginsof none are known.The latest paper relating to the evolution of bioluminescence(4) focuses on the most common chemistry causing biolumines-cence in marine organisms – coelenterazine. This wasdiscovered in the coelenterate jellyfish Aequorea by NobelLaureate Osamu Shimomura (6,7). Ironically, this may be amisnomer, because luminous jellyfish do not appear to be ableto synthesize coelenterazine de novo, and therefore must obtainit through the food chain. Coelenterazine is predicted to besynthesized from the cyclization of three amino acids – Phe,Tyr, Tyr – which is not a problem via random mutation, refutingHoyle’s argument (3). The most credible evolutionary origin forcoelenterazine is as an oxygen metabolite scavenger analogousto vitamin C in our blood. But the puzzle for the molecularbiologist, highlighted in this latest paper (4), is that when theknown amino acid sequences for coelenterazine luciferasesand photoproteins are compared there is very little similaritybetween them, even within quite closely related phylogeneticgroups. So how could such diversity in protein sequencescatalyse the same chemical reaction to produce light?Vassel et al. used albumin as a model for a primeval luciferase(4), because it is known to bind a wide range of substances,and was predicted to form a solvent cage that could providethe necessary electrochemical environment to catalysecoelenterazine chemiluminescence. The results show, for thefirst time, that albumin does indeed catalyse coelenterazinechemiluminescence, constistent with a mono-oxygenaseenzymatic activity. Evidence for this being truly enzymatic wasbased on the fact that heat denaturation destroyed the activity,it was saturable by the substrate coelenterazine, it increasedwith alkaline pH, and it was blocked by several divalent cationsand by drugs known to bind to albumin. Using modellingsoftware, the coelenterazine binding site was identified asSudlow site 1. Only drugs that bound this site inhibited the

In the last years, intracellular organella have emerged as key components in the generation and transduction of Ca(2+) signals in adrenal chromaffin cells. Therefore, accurate measurements of Ca(2+) inside cytoplasmic organella are essential for a comprehensive analysis of the Ca(2+) redistribution that follows cell stimulation. We have engineered the Ca(2+)-sensitive photoprotein aequorin to monitor selectively Ca(2+) within the endoplasmic reticulum and the mitochondria. The targeted aequorins were delivered to the appropriate organelles of bovine chromaffin cells by using a herpes simplex virus-based amplicon vector, which permits efficient gene transfer and high levels of expression in infected cells. We have investigated the relationship between the caffeine and InsP(3)-sensitive Ca(2+) pools and the presence of the Ca(2+)-induced Ca(2+) release (CICR) mechanism in chromaffin cells. We find that ER Ca(2+) pools responding to caffeine and to InsP(3) mostly overlap and that CICR can be induced by Ca(2+) entry elicited by high K(+) depolarization. Moreover, the activation of Ca(2+) channels, either the voltage-gated Ca(2+) channels on the plasma membrane or the channels on the endoplasmic reticulum (ER), generates subplasmalemmal high [Ca(2+)](c) domains that induce Ca(2+) uptake by mitochondria. Interestingly, only a subpopulation of mitochondria, the one contained in the pool located close to the plasma membrane and the ryanodine receptors, take up Ca(2+) efficiently, and the [Ca(2+)](M) reaches values of 300-500 micro M.

Creatures that glow have mesmerised people for aeons. During the last few hundred years, scientists have been trying to reveal exactly how organisms produce this light, or bioluminescence. It has been estimated that approximately 30 or more chemically distinct bioluminescence systems have evolved independently. The luciferase enzymes that catalyse bioluminescent reactions use a variety of different structures to produce light. This article looks at the X-ray crystal structures known for five different types of luciferases: bacterial, dinoflagellate, firefly, and two classes of coelenterates (anthozoan and hydrozoan). The structures of these enzymes reveal details of how they catalyse bioluminescence reactions, and their remarkable diversity of structure, mechanism and substrate specificity. Two accessory fluorescent proteins are also described. Key Concepts: Luciferases are enzymes that catalyse reactions that produce bioluminescence. Approximately 30 distinct bioluminescent systems have evolved. Bioluminescent reactions all involve oxygenation of a substrate to generate a peroxide intermediate, which then breaks down to give an electronically excited product that emits light. X-ray crystallographic structures are known for the luciferases in five different bioluminescent systems: bacteria, dinoflagellates, fireflies, hydrozoa and anthozoa. Each of these luciferases has an entirely different molecular structure and catalyzes bioluminescence in a unique way. Keywords: luciferase; luciferin; bioluminescence; chemiluminescence; antenna proteins; green fluorescent protein; yellow fluorescent protein; photoprotein; bacterial luciferase; firefly luciferase; dinoflagellate luciferase; renilla luciferase; aequorin; obelin; clytin; hydrozoa; anthozoa

Mutation of the Trp92 that is known to lie within the active site of the photoprotein obelin from Obelia longissima , results in a shift of the bioluminescence color from blue ( λ max =485 nm) to violet. The corrected spectrum shows a new band with λ max =410 nm now contributing equally to the one at longer wavelength. The crystal structure of this W92F obelin determined at 1.72 A resolution shows that there is no significant change in the dimensions of the active site between WT obelin (recombinant Ca 2+ -regulated photoprotein from Obelia longissima ) and the mutant. It is proposed that the bioluminescence spectral shift results from removal of a hydrogen bond from the indole of W92 nearby a hydroxyl belonging to the 6-phenyl substituent of the substrate coelenterazine. Propagation of this change through a conjugated bond system in the excited state of the product coelenteramide affects the coupling of the N1-position and the hydrogen-bonded Y138.

In the last decade, the study of Ca2+ homeostasis within organelles in living cells has been greatly enhanced by the utilisation of a recombinant Ca(2+)-sensitive photoprotein, aequorin. Aequorin is a Ca2+ sensitive photoprotein of a coelenterate that, in the past, was widely employed to measure Ca2+ concentration in living cells. In fact, the purified protein was widely used to monitor cytoplasmic [Ca2+] changes in invertebrate muscle cells after microinjection. However, due to the time-consuming and traumatic procedure of microinjection, the role of aequorin in the study of Ca2+ homeostasis remained confined to a limited number of cells (giant cells) susceptible to microinjection. Thus, in most instances, it was replaced by the fluorescent indicators developed by Roger Tsien and coworkers. The cloning of aequorin cDNA [Inouye et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:3154-3158] and the explosive development of molecular biology offered new possibilities in the use of aequorin, as microinjection has been replaced by the simpler technique of cDNA transfection. As a polypeptide, aequorin allows the endogenous production of the photoprotein in cell systems as diverse as bacteria, yeast, slime molds, plants, and mammalian cells. Moreover, it is possible to specifically localise it within the cell by including defined targeting signals in the amino acid sequence. Targeted recombinant aequorins represent to date the most specific means of monitoring [Ca2+] in subcellular organelles. In this review, we will not discuss the procedure of aequorin microinjection and its use as purified protein but we will present the new advances provided by recombinant aequorin in the study of intracellular Ca2+ homeostasis, discussing in greater detail the advantages and disadvantages in the use of this probe.

Over recent decades, diverse intracellular organelles have been recognized as key determinants of Ca(2+) signaling in eukaryotes. In yeast however, information on intra-organellar Ca(2+) concentrations is scarce, despite the demonstrated importance of Ca(2+) signals for this microorganism. Here, we directly monitored free Ca(2+) in the lumen of the endoplasmic reticulum (ER) of yeast cells, using a specifically targeted version of the Ca(2+)-sensitive photoprotein aequorin. Ca(2+) uptake into the yeast ER displayed characteristics distinctly different from the mammalian ER. At steady-state, the free Ca(2+) concentration in the ER lumen was limited to approximately 10 microM, and ER Ca(2+) sequestration was insensitive to thapsigargin, an inhibitor specific for mammalian ER Ca(2+) pumps. In pmr1 null mutants, free Ca(2+) in the ER was reduced by 50%. Our findings identify the secretory pathway pump Pmr1, predominantly localized in the Golgi, as a major component of ER Ca(2+) uptake activity in yeast.

The first part of this survey focuses on immunoassays and related ligand:binder assays (receptor:ligand, DNA probe) that use either a luciferase or a photoprotein as a label. In addition, references to assays that use a conventional label detected using a bioluminescent assay are included. The second part of the survey collects together references to publications on recombinant fusion proteins in which one of the fused proteins is bioluminescent (e.g., a luciferase or a photoprotein). References are cited by year and then alphabetically by first author. Copyright © 1999 John Wiley & Sons, Ltd.

An experiment was proposed and accomplished that was based on the hypothesis of the dissociation of the luciferase-luciferin complex in photoexcitation. A pump-probe experiment was performed with the use of picosecond laser pulses and was based on the effect of quenching of enzyme tryptophan fluorescence caused by luciferin binding. A photoinduced increase of the tryptophan fluorescence intensity was detected. Experimental results were interpreted on the basis of the assumptions on photoinduced dissociation of the luciferin-luciferase complex and Forster energy transfer from tryptophan to luciferin. Under the assumption on the photoinduced dissociation and stationary quenching of tryptophan fluorescence the rate of propagation of the conformational changes in the protein caused by the complex dissociation was estimated to be >20 m/s.

It was previously assumed that E·LH2-AMP was the intermediate complex in the synthesis of Ap4A catalyzed by firefly luciferase (EC 1.13.12.7), when luciferin (LH2) was used as cofactor. However, here we show that LH2 is partly transformed, shortly after the onset of the luciferase reaction, to dehydroluciferin (L) with formation of an E·L-AMP complex which is the main intermediate for the synthesis of Ap4A. Formation of three more derivatives of LH2 were also observed, related to the production of light by the enzyme. CoA, a known stimulator of light production, inhibits the synthesis of Ap4A by reacting with the E·L-AMP complex and yielding L-CoA.

1. In smooth muscle, both Ca2+ release from the sarcoplasmic reticulum (SR) and Ca2+ influx across the plasma membrane are responsible for the increase in the cytosolic Ca2+ level ([Ca2+]i). To understand further the role of SR on smooth muscle contraction, the effects of an inhibitor of the SR Ca2+ pump, cyclopiazonic acid (CPA 10 microM), an inhibitor of the Ca(2+) -induced Ca2+ release, ryanodine, (10 microM), and an activator of the Ca(2+) -induced Ca2+ release, caffeine (20 mM), on [Ca2+]i and contractile force were examined in the ferret portal vein loaded with a photoprotein, aequorin. 2. CPA induced a small increase in the aequorin signal reaching a maximum at 7 min. Several minutes after the increase in the aequorin signal, muscle tension increased reaching a maximum at 21.5 min. In contrast, ryanodine changed neither the aequorin signal nor contraction. In the presence of ryanodine, caffeine induced a sustained increase in the aequorin signal and transient contraction. After washing ryanodine and caffeine, the aequorin signal and muscle tone returned to their respective control levels. After treatment with ryanodine and caffeine, the second addition of caffeine was almost ineffective whereas CPA still increased the aequorin signal and muscle tension. 3. In the presence of external Ca2+, noradrenaline (NA, 10 microM) induced a transient increase followed by a sustained increase in the aequorin signal and sustained contraction. In contrast, KCl (70 mM) induced sustained increases in the aequorin signal and sustained contraction. In Ca(2+) -free solution, NA induced a small transient increase in the aequorin signal and a small transient contraction. These changes were inhibited in the presence of CPA or on pretreatment of the muscle with ryanodine and caffeine. These results suggest that CPA or ryanodine and caffeine depleted Ca2+ in SR. High K+ was ineffective in the absence of external Ca2+. 4. In the presence of external Ca2+ and CPA, NA and high K+ induced larger aequorin signals than in the absence of CPA, whereas the magnitude and shape of the contractions did not change. In contrast, pretreatment with ryanodine and caffeine did not have such an effect. In the muscle pretreated with ryanodine and caffeine, CPA changed the responses to high K+ and NA in a similar manner to that in the muscle without the pretreatment with ryanodine and caffeine. 5. Dissociation of contraction from [Ca2+]i as measured with aequorin suggests that NA and high K+ increase Ca2+ in two compartments: a compartment containing contractile elements (contractile compartment) and another compartment unrelated to contractile elements (non-contractile compartment). Because CPA augmented the stimulant-induced increase in aequorin signal without changing contraction, the non-contractile compartment may be located near the SR and the CPA-sensitive SR Ca2+ pump may regulate the Ca2+ level in this compartment. However, because CPA changed neither the magnitude nor shape of the contractions in the presence of external Ca2+, the SR Ca2+ pump may have little effect on regulation of Ca2+ level in the contractile compartment. Furthermore, the release of Ca2+ from SR seems to have little effect on the increase in the contractile Ca2+ because ryanodine and caffeine changed neither the aequorin signals nor contractions induced by NA and high K+ in the presence of external Ca2+ in the ferret portal vein.

Isolated characean internodal cells of Nitellopsis obtusa can be stored in artificial pond water for many days, but they cannot survive in 10 mol m -3 NaCl solution unless more than several mol m -3 Ca 2+ is added. Short-term effects of NaCI stress on the cytosolic concentration of Ca 2+ ([Ca 2+ ] c )F cytosolic pH (pH c ) and vacuolar pH (pH v ) were studied in relation to the external concentration of Ca 2+ ([Ca 2+ ] e ). Changes in [Ca 2+ ] c were measured with light emission from a Ca 2+ -sensitive photoprotein, semisynthetic fch-aequorin which had been injected into the cytosol. Both pH c and pH v were measured with double-barrelled pH-sensitive microelectrodes. When internodal cells were treated with 100 mol m -3 NaCl (0.1 mol m -3 [Ca 2+ ] e ), [Ca 2+ ] c increased and then recovered to the original level within 60 min. The time course of the transient change in [Ca 2+ ] c was not influenced by the level of [Ca 2+ ] e (0.1 and 10 mol m -3 ). In some cases, the transient increase in [Ca 2+ ] c was induced only by increasing external osmotic pressure with sorbitol. In response to treatment with 100 mol m -3 NaCl (0.1 mol m -3 [Ca 2+ ] e ), pH c decreased by 0.1-0.2 units after 10 min but recovered after 30-60 min, while pH v increased by 0.4-0.5 units after 2-50 min and tended to recover after 60 min. The initial changes in both pH c and pH v were suppressed when [Ca 2+ ] e was raised from 0.1 to 10 mol m -3 . These results show that the charophyte alga Nitellopsis can regulate [Ca 2+ ] c , pH c and pH v under NaCI stress in the short term and that the protective effect of Ca 2+ on salinity stress is apparently unrelated to perturbation of Ca 2+ and pH homeostasis.

In order to improve calibration of firefly luciferase signals obtained by injecting the enzyme into single, isolated heart and liver cells we have investigated why the luminescence from cells is greatly depressed compared with in vitro (in mammalian ionic milieu) and why the decay of the intracellular signal is remarkably slow. We have shown that inorganic pyrophosphatase greatly depresses the signal in vitro and that micromolar concentrations of inorganic pyrophosphate, comparable with that in cytoplasm, reverse this inhibition and stabilize the signal, eliminating its decay. Higher concentrations of pyrophosphate depress the signal by inhibiting ATP-binding to luciferase. Luciferase-injected cells exposed to extracellular luciferin concentrations above about 100 mumol/l (corresponding to a cytoplasmic level of c. 5-10 mumol/l because of a transplasmalemmal gradient) show a gradual, irreversible loss of signal. We attribute this phenomenon (which is not seen in vitro) to the gradual accumulation of a luminescently inactive, irreversible, luciferase-oxyluciferin complex. At low luciferin levels this complex is prevented from forming by cytoplasmic pyrophosphate. Above c. 100 mumol/l extracellular luciferin, the pyrophosphate level in the cytoplasm fails to fully prevent the complex forming. In vitro this phenomenon does not occur because the luciferase concentrations and hence oxyluciferin levels are orders of magnitude lower than in cells injected with concentrated luciferase solutions, which have a cytoplasmic luciferase concentration of approximately 2-4 mumol/l.

Activation of nuclear transcription factors, breakdown of nuclear envelope and apoptosis represent a group of nuclear events thought to be modulated by changes in nucleoplasmic Ca2+ concentration, [Ca2+]n. Direct evidence for, or against, this possibility has been, however, difficult to obtain because measurements of [Ca2+]n are hampered by major technical problems. Here we describe a new approach for selectively monitoring Ca2+ concentrations inside the nucleus of living cells, which is based on the construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and a nuclear translocation signal derived from the rat glucocorticoid receptor. This modified aequorin (nuAEQ), stably expressed in HeLa cells, was largely confined to the nucleoplasm and thus utilized for monitoring [Ca2+]n in intact cells. No significant differences were observed between [Ca2+]n and cytosolic Ca2+ concentration ([Ca2+]i) under resting conditions. Upon stimulation of surface receptors linked to inositol-1,4,5-trisphosphate (InsP3) generation, and thus to intracellular Ca2+ signalling, the kinetics of [Ca2+]i and [Ca2+]n increases were indistinguishable. However, for the same rise in [Ca2+]i, the amplitude of [Ca2+]n increase was larger when evoked by Ca2+ mobilization from internal stores than when induced by Ca2+ influx across the plasma membrane. The functional significance of these transient nucleus-cytosol Ca2+ gradients is discussed.

The luminescence of obelin is initiated by NaOCl in a reaction mixture containing no calcium. The addition of Mn2+ enhances the light emission > 300-fold. Sodium azide and histidine, as singlet oxygen quenchers, inhibit NaOCl-activated obelin luminescence in the presence or absence of Mn2+. This suggests that the addition of NaOCl to the mixture causes singlet oxygen formation (stimulated by Mn2+ ions), and singlet oxygen initiates the light-emitting reaction.

An M-type intermediate is formed in the 13- cis -bR photocycle in purple membranes at high pH. This is presumably due to deprotonation of the same group whose deprotonation causes a large increase in rate of M formation in the trans -bR photocycle (the ‘alkaline transition’). For Triton X-100-solubilized bR, the alkaline transition is shifted to a lower pH value by more than 2 pH units. The alkaline transition in Triton-solubilized preparations changes the efficiency of the M intermediate formation in the 13- cis -sbR photocycle. The M intermediate formation in 13- cis -sbR, as in the case of trans -sbR, is completely inhibited when the blue ‘acidic’ bR is formed at low pH. The protonation state of the group affecting formation of the M intermediate in 13- cis -bR at high pH and the group which is responsible for the transition to the blue acidic form influence in a similar way the equilibrium between bR isomers in the dark-adapted form as well as the rate of dark adaptation.

Surface bound pH indicators were applied to study the proton transfer reactions in the mutant Asp85-->Glu of bacteriorhodopsin in the native membrane. The amino acid replacement induces a drastic acceleration of the overall rise of the M intermediate. Instead of following this acceleration, proton ejection to the extracellular membrane surface is not only two orders of magnitude slower than M formation, it is also delayed as compared with the wild-type. This demonstrates that Asp85 not only accepts the proton released by the Schiff's base but also regulates very efficiently proton transfer within the proton release chain. Furthermore, Asp85 might be the primary but is not the only proton acceptor/donor group in the release pathway. The Asp85-->Glu substitution also affects the proton reuptake reaction at the cytoplasmic side, although Asp85 is located in the proton release pathway. Proton uptake is slower in the mutant than in the wild-type and occurs during the lifetime of the O intermediate. This demonstrates a feed-back mechanism between Asp85 and the proton uptake pathway in bacteriorhodopsin.

We have measured the current generated by light-activated proton release from bacteriorhodopsin into solution as a function of both pH and ionic strength. We find that proton release into solution decreases with increasing pH with an intrinsic p K a of 8.2 ± 0.2. This pH dependence indicates that the deprotonation of a certain group inhibits or abolishes proton release. Under physiological conditions, this group either releases a proton directly into solution or interacts with the site of proton release. The most immediate candidates for this protonatable species are tyrosine-57, tyrosine-185, arginine-82, and water; acting individually or cooperatively. The salt dependence of the apparent p K a of this group also allows us to calculate the surface charge density of about −5 charges per bacteriorhodopsin, compatible with previous estimates.

The H(+)-ATPase from the plasma membrane of Saccharomyces cerevisiae was isolated and purified. The enzyme was reconstituted with bacteriorhodopsin into asolectin liposomes by detergent dialysis at a molar ratio of 1 H(+)-ATPase to 50 bacteriorhodopsins. The overall orientation of the proteins is such that proton pumping to the vesicle interior occurs upon illumination and after addition of ATP. All liposomes which contain H(+)-ATPase also contain bacteriorhodopsin. The rate of ATP hydrolysis was measured as function of pH in the dark and during illumination of the proteoliposomes. The pH dependency can be described by the protonation of a monovalent group from the outside with an apparent pK of 7.3 and the deprotonation of a monovalent group at the inside with an apparent pK of 3.7. Inside and outside refer to the orientation of the H(+)-ATPase in the liposomes which is opposite to that occurring in vivo. It is concluded that the first step in the reaction cycle is the binding of a proton from the cytosol which is followed by ATP binding, ATP hydrolysis on the enzyme and the release of ADP and phosphate, and finally the proton is released from the enzyme into the external medium.

A protocol for the separation of phosphorhodopsin from phospho-opsin has been developed. The method takes advantage of the finding that, while 0.5% N,N-dimethyldodecylamine-N-oxide completely solubilises membrane-embedded phosphorhodopsin, at this concentration of detergent, phospho-opsin is only sparingly soluble. Phosphorhodopsin solubilised in this manner may be freed from contaminant phospho-opsin by chromatography on hydroxyapatite. Using this method, the rhodopsin-kinase-catalysed phosphorylation of photoexcited rhodopsin and native rhodopsin was studied in rod outer-segment membranes at different levels of bleaching. Prior to analysis of the phosphorylation mixture, the phosphorylated form of photoexcited rhodopsin was converted into phospho-opsin by treatment with NH2OH. It was found that, while at a 5% bleach level the amount of phosphorhodopsin produced was 15% that of phospho-opsin, at 60% bleaching the phosphorhodopsin was less than 1% of phospho-opsin. The phosphorylation reaction under different bleaching conditions was also studied in a completely soluble system (using 2% dodecyl maltoside) and the pattern of phosphate incorporation into rhodopsin versus opsin was identical to that in the membrane system. We have previously proposed that rhodopsin kinase normally exists in an inactive form and is only activated following interaction with photoexcited rhodopsin. The present work strengthens this conclusion and also shows that, following activation, the kinase preferentially phosphorylates photoexcited rhodopsin but can also act upon unbleached rhodopsin. Two possible mechanisms for the activation of the kinase are considered. From the distribution of phosphorhodopsin and phospho-opsin at different bleaching levels, the relative rates of the phosphorylation of photoexcited rhodopsin (kR*) and rhodopsin (kR) were calculated. kR*/kR values for the membrane system of 71 +/- 20 and, for the solubilised system, of 80 +/- 19 were obtained. The algebraic equation used to obtain these values highlights the fact that the ratio of the concentrations of the two substrates, photoexcited rhodopsin and rhodopsin, in a sample, determines the final distribution of phosphate between bleached and unbleached rhodopsin. This conclusion may contribute to the understanding of 'high-gain' phosphorylation observed previously.

Crystals of recombinant aequorin, the photoprotein from the jellyfish Aequorea victoria, have been grown from solutions containing sodium phosphate. The crystals grow as thin plates which diffract to beyond 2.2 A resolution. The crystals are orthorhombic, space group P21212 1; the axes are a = 89.1(1), b = 88.4(1), and c = 52.7(1) A. The asymmetric unit contains two molecules. Crystals exposed to calcium ion solutions emit a steady glow and slowly deteriorate, confirming that the crystals consist of a charged, competent photoprotein. This represents the first successful preparation of single crystals of a photoprotein suitable for diffraction analysis. © 1993 Wiley-Liss, Inc.

In the photoprotein of an Okinawan squid bioluminescence of Symplectoteuthis oualaniensis L a dehydrocoelenterazine has been assigned as a chromophoric precursor to its apoprotein. To prove this mechanism, we have established new synthetic route to ca. 100 %- 13 C incorporated dehydrocoelenterazine and coelenterazine at the neighboring carbon of the 2-position of 2,3-dihydroimidazo-[1,2a]-pyrazinone skeleton. This 13 C enriched dehydrocoelenterazine readily converted in equilibrium between its adduct forms as a diastereomixture with glutathione (GSH) or dithiothreitol (DTT) compounds having sulfhydryl group. Structures of such adducts were fixed under acidic conditions and then discussed by NMR spectroscopy as well as absorbance and fluorescence spectra.