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
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
An automated aequorin luminescence-based functional calcium assay for G-protein-coupled receptors.
AuthorsUngrin MD, Singh LM, Stocco R, Sas DE, Abramovitz M
JournalAnal Biochem
PubMed ID10405290
We describe in detail an automated and highly sensitive functional assay for calcium-coupled receptors (those receptors whose activation results in an increase in intracellular calcium levels) utilizing coelenterazine-charged aequorin as a probe for intracellular calcium levels ([Ca(2+)](i)). The assay was originally established to investigate Galpha(q)-coupled prostanoid receptors, which are members ... More
N-methyl-D-aspartate receptors expressed in a nonneuronal cell line mediate subunit-specific increases in free intracellular calcium.
AuthorsGrant ER, Bacskai BJ, Pleasure DE, Pritchett DB, Gallagher MJ, Kendrick SJ, Kricka LJ, Lynch DR
JournalJ Biol Chem
PubMed ID8995308
N-methyl-D-aspartate (NMDA) receptors can mediate cell death in neurons and in non-neuronal cells that express recombinant NMDA receptors. In neurons, increases in intracellular calcium correlate with NMDA receptor-mediated death, supporting a key role for loss of cellular calcium homeostasis in excitotoxic cell death. In the present study, free intracellular calcium ... 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
Reorientation of seedlings in the earth's gravitational field induces cytosolic calcium transients.
AuthorsPlieth C, Trewavas AJ
JournalPlant Physiol
PubMed ID12068119
The gravitational field controls plant growth, morphology, and development. However, the underlying transduction mechanisms are not well understood. Much indirect evidence has implicated the cytoplasmic free calcium concentration ([Ca(2+)](c)) as an important factor, but direct evidence for changes in [Ca(2+)](c) is currently lacking. We now have made measurements of [Ca(2+)](c) ... More