Stable expression of a synthetic gene for the human motilin receptor: use in an aequorin-based receptor activation assay.
AuthorsCarreras CW, Siani MA, Santi DV, Dillon SB
JournalAnal Biochem
PubMed ID11779105
'A synthetic gene for the human motilin receptor containing 33 unique restriction sites was designed and stably coexpressed in HEK293 cells with the bioluminescent Ca(2+) indicator protein aequorin. The dose-dependent response of the receptor to motilin was demonstrated using transient transfections, and a stable cell line was selected. [(125)I]Motilin binding ... More
The use of Renilla luciferase, Oplophorus luciferase, and apoaequorin as bioluminescent reporter protein in the presence of coelenterazine analogues as substrate.
AuthorsInouye S, Shimomura O
JournalBiochem Biophys Res Commun
PubMed ID9144537
'To investigate the use of various luciferases as reporter protein, the substrate specificity of recombinant Renilla luciferase, Oplophorus luciferase and recombinant apoaequorin was examined using 23 kinds of coelenterazine analogues as the substrate. The intensity of luminescence was generally highest with Oplophorus luciferase and lowest with apoaequorin, but varied widely ... More
Characterization of coelenterazine analogs for measurements of Renilla luciferase activity in live cells and living animals.
AuthorsZhao H, Doyle TC, Wong RJ, Cao Y, Stevenson DK, Piwnica-Worms D, Contag CH
JournalMol Imaging
PubMed ID15142411
'In vivo imaging of bioluminescent reporters relies on expression of light-emitting enzymes, luciferases, and delivery of chemical substrates to expressing cells. Coelenterazine (CLZN) is the substrate for a group of bioluminescent enzymes obtained from marine organisms. At present, there are more than 10 commercially available CLZN analogs. To determine which ... More
Oscillations in free [Ca2+]i during early cell division cycles in Xenopus laevis embryos.
AuthorsMiller B, Chauhan A, Jaffe LF, Miller AL
JournalBiol Bull
PubMed ID7811797
Preparation and handling of aequorin solutions for the measurement of cellular Ca2+.
AuthorsShimomura O
JournalCell Calcium
PubMed ID1959127
Main characteristics of the various types of aequorin presently available for measuring cellular Ca2+, i.e. heterogeneous aequorin, isoaequorins, recombinant aequorin, fluorescein-labeled aequorin and semi-synthetic aequorins, are summarized. Basic techniques of preparing and handling the solutions of those aequorins for measuring Ca2+, including such techniques as concentrating aequorin solutions, freeze-drying, changing ... More
Light-emitting properties of recombinant semi-synthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium.
AuthorsShimomura O, Musicki B, Kishi Y, Inouye S
JournalCell Calcium
PubMed ID8519061
15 kinds of recombinant semi-synthetic aequorins and a recombinant fluorescein-conjugated aequorin were prepared and their properties in Ca(2+)-triggered luminescence were studied. The semi-synthetic aequorins showed a wide range of Ca(2+)-sensitivity. The luminescence intensity of a high-sensitivity type (hcp-aequorin) was greater than 10(4)-times that of a low-sensitivity type (n-aequorin) at pCa ... More
Membrane permeability of coelenterazine analogues measured with fish eggs.
AuthorsShimomura O
JournalBiochem J
PubMed ID9291095
To determine the suitability of various coelenterazine analogues for the regeneration of aequorin in living cells, the membrane permeabilities of 11 analogues were measured using the eggs of the killifish Fundulus grandis by soaking the eggs in solutions containing the analogues. The results indicated that e-coelenterazine, which has an exceptionally ... More
The relative rate of aequorin regeneration from apoaequorin and coelenterazine analogues.
AuthorsShimomura O, Kishi Y, Inouye S
JournalBiochem J
PubMed ID8280050
The regeneration of an active semi-synthetic aequorin, from apoaequorin produced in cells and a coelenterazine analogue, is a key step in measuring Ca2+ in the cells. The relative rates of the regeneration of semi-synthetic aequorins from apoaequorin and 28 synthetic coelenterazine analogues were compared. The results indicated that the rate ... More
Semi-synthetic aequorins with improved sensitivity to Ca2+ ions.
AuthorsShimomura O, Musicki B, Kishi Y
JournalBiochem J
PubMed ID2803250
Thirty-seven coelenterazine analogues were synthesized and incorporated into apo-aequorin, yielding 30 semi-synthetic aequorins that have the capacity to emit a significant amount of light in the presence of Ca2+. The properties of resultant photoproteins were investigated. The most prominent feature of those photoproteins was the wide range in their sensitivities ... More
Imaging of multicellular large-scale rhythmic calcium waves during zebrafish gastrulation.
AuthorsGilland E, Miller AL, Karplus E, Baker R, Webb SE
JournalProc Natl Acad Sci U S A
PubMed ID9874788
Oscillations of cytosolic free calcium levels have been shown to influence gene regulation and cell differentiation in a variety of model systems. Intercellular calcium waves thus present a plausible mechanism for coordinating cellular processes during embryogenesis. Herein we report use of aequorin and a photon imaging microscope to directly observe ... More
Imaging reversal of multidrug resistance in living mice with bioluminescence: MDR1 P-glycoprotein transports coelenterazine.
AuthorsPichler A, Prior JL, Piwnica-Worms D
JournalProc Natl Acad Sci U S A
PubMed ID14755051
Coelenterazine is widely distributed among marine organisms, producing bioluminescence by calcium-insensitive oxidation mediated by Renilla luciferase (Rluc) and calcium-dependent oxidation mediated by the photoprotein aequorin. Despite its abundance in nature and wide use of both proteins as reporters of gene expression and signal transduction, little is known about mechanisms of ... More
Intracellular free calcium oscillations in normal and cleavage-blocked embryos and artificially activated eggs of Xenopus laevis.
AuthorsKeating TJ, Cork RJ, Robinson KR
JournalJ Cell Sci
PubMed ID7983182
We have measured levels of intracellular free calcium ([Ca2+]i) in albino Xenopus laevis embryos using recombinant aequorin and a photon-counting system. We observed sinusoidal oscillations in [Ca2+]i that had the same frequency as cleavage, with cleavage occurring when [Ca2+]i was lowest. An increase in calcium was seen to precede first ... More
Cause of spectral variation in the luminescence of semisynthetic aequorins.
AuthorsShimomura O
JournalBiochem J
PubMed ID7887908
Aequorin emits light in the presence of Ca2+, decomposing into apoaequorin, coelenteramide and CO2. Semisynthetic aequorins, produced by replacing the coelenterazine moiety in aequorin with analogues of coelenterazine, showed widely different sensitivities to Ca2+ as well as certain spectral variations. A group of semisynthetic aequorins, e-type aequorins, showed bimodal luminescence, ... More
Transduction of intracellular calcium signals through G protein-mediated activation of phospholipase C by recombinant sphingosine 1-phosphate receptors.
AuthorsAn S, Bleu T, Zheng Y
JournalMol Pharmacol
PubMed ID10220556
Sphingosine 1-phosphate (S1P) increases intracellular Ca2+ concentration in many cell types, but the signaling mechanism remains uncertain. The recent identification of three closely related seven-transmembrane domain receptors for S1P, termed Edg1, H218, and Edg3, support the extracellular ligand role of S1P and allowed examination of Ca2+ responses mediated specifically by ... More
Recombinant human G protein-coupled lysophosphatidic acid receptors mediate intracellular calcium mobilization.
AuthorsAn S, Bleu T, Zheng Y, Goetzl EJ
JournalMol Pharmacol
PubMed ID9804623
Mobilization of intracellular Ca2+ is a critical cellular response to lysophosphatidic acid (LPA) in many cell types. Recent identification of endothelial differentiation gene (Edg) 2 and Edg4 as subtypes of G protein-coupled receptors for LPA allowed examination of the Ca2+ mobilization mediated specifically by each subtype. To reduce endogenous background ... More