Free calcium pulses following fertilization in the ascidian egg.
AuthorsSpeksnijder JE, Corson DW, Sardet C, Jaffe LF
JournalDev Biol
PubMed ID2767333
'Using the calcium-specific, chemiluminescent photoprotein aequorin, we have measured changes in the concentration of free cytosolic calcium at fertilization in single eggs of the ascidians Phallusia mammillata and Ciona intestinalis. Shortly after insemination, the free calcium concentration rises within a minute from a resting level of about 90 nM in ... More
Focal [Ca2+]i increases detected by aequorin but not by fura-2 in histamine- and caffeine-stimulated swine carotid artery.
AuthorsRembold CM, Van Riper DA, Chen XL
JournalJ Physiol
PubMed ID8576847
'1. We hypothesized that the homogeneity of intracellular [Ca2+] ([Ca2+]i) varies and is regulated in arterial smooth muscle. 2. We evaluated this hypothesis by exploiting the different characteristics of several [Ca2+]i indicators: (1) aequorin, which theoretically can measure focal increases in [Ca2+]i, (2) fura-2, which is predominantly a measure of ... More
The part played by inositol trisphosphate and calcium in the propagation of the fertilization wave in sea urchin eggs.
AuthorsSwann K, Whitaker M
JournalJ Cell Biol
PubMed ID3491080
'Sea urchin egg activation at fertilization is progressive, beginning at the point of sperm entry and moving across the egg with a velocity of 5 microns/s. This activation wave (Kacser, H., 1955, J. Exp. Biol., 32:451-467) has been suggested to be the result of a progressive release of calcium from ... More
Developmental regulation of IgM secretion: the role of the carboxy-terminal cysteine.
AuthorsSitia R, Neuberger M, Alberini C, Bet P, Fra A, Valetti C, Williams G, Milstein C
JournalCell
PubMed ID2107027
'B lymphocytes do not secrete IgM, and plasma cells only secrete IgM polymers. Here we show that both events are attributable to the tailpiece found at the carboxyl terminus of mus chains, and we specifically implicate Cys-575. Thus, if Cys-575 was mutated, IgM was secreted by B cells. Similarly, a ... More
Halothane alters cytosolic calcium transient in tracheal smooth muscle.
AuthorsJones KA, Housmans PR, Warner DO, Lorenz RR, Rehder K
JournalAm J Physiol
PubMed ID8338185
'Halothane is a potent bronchodilator that attenuates vagally mediated constriction of canine airways, in part by a direct action on the smooth muscle cell. In tracheal smooth muscle, acetylcholine (ACh) produces a transient peak increase in cytosolic ionized calcium concentration ([Ca2+]i), which declines to a sustained plateau concentration that is ... More
Video light microscopic imaging of the calcium signal that initiates nuclear envelope breakdown in sand dollar (Echinaracnius parma) cells.
AuthorsSilver R
JournalBiol Bull
PubMed ID7811794
Structure of the light-emitting moiety of aequorin.
AuthorsShimomura O, Johnson FH
JournalBiochemistry
PubMed ID4402156
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
Imaging [Ca2+]i with aequorin using a photon imaging detector.
AuthorsMiller AL, Karplus E, Jaffe LF
JournalMethods Cell Biol
PubMed ID8201982
Adenine dinucleotide-mediated cytosolic free Ca2+ oscillations in single hepatocytes.
Single rat hepatocytes microinjected with aequorin respond to Ca(2+)-mobilizing agonists, including ADP and ATP, with oscillations in cytosolic free Ca2+. We show here that single rat hepatocytes also respond to the adenine dinucleotides Ap3A and Ap4A with Ca2+ oscillations which resemble those induced by ADP and ATP. ... More
Quenching of biotinylated aequorin bioluminescence by dye-labeled avidin conjugates: application to homogeneous bioluminescence resonance energy transfer assays.
AuthorsAdamczyk M, Moore JA, Shreder K
JournalOrg Lett
PubMed ID11405714
[see reaction]. Avidin conjugates containing the covalently attached dyes QSY-7 and dabcyl were prepared and shown to quench the bioluminescence of biotinylated aequorin. Quenching efficiency was shown to be dependent on both the label-to-avidin ratio and the concentration of the avidin conjugate. These properties were exploited to develop a homogeneous ... More
Calibration of micropipets using the bioluminescent protein aequorin.
AuthorsGrosvenor AL, Crofcheck CL, Anderson KW, Scott DL, Daunert S
JournalAnal Chem
PubMed ID9253255
A new method for calibrating micropipets and determining accurate injection volumes using a pressure-based injector has been developed. This method employs the bioluminescent protein aequorin and can be used to determine injection volumes as small as 3 pL. The calibration plots are linear over at least 3 orders of magnitude. ... More
Recombinant aequorin as a probe for cytosolic free Ca2+ in Escherichia coli.
AuthorsKnight MR, Campbell AK, Smith SM, Trewavas AJ
JournalFEBS Lett
PubMed ID2037058
We describe a novel and simple method for the measurement of bacterial cytosolic free calcium ([Ca2+]i) using recombinant aequorin reconstituted within live bacterial cells. Using this method we have measured the effects of external calcium, complement, phagocytosis and antibiotics on the [Ca2+]i of Escherichia coli. In principle this method should ... 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
Measurement of cytosolic free calcium in mammalian cells with aequorin.
AuthorsSnowdowne KW, Borle AB
JournalAm J Physiol
PubMed ID6093570
Measurement of cytosolic free calcium (Cai) in small mammalian cells has been achieved by incorporating into cells the photoprotein aequorin by hypoosmotic shock treatment (HOST). The method and the instrumentation necessary for monitoring Cai are described in detail. We present evidence that the aequorin-Ca light signal originates in the cytosol ... More
Calcium transients in single adrenal chromaffin cells detected with aequorin.
The effect of 55 mM K+ and nicotine on intracellular free calcium was monitored in bovine adrenal chromaffin cells microinjected with aequorin. In contrast to results with quin 2, which suggested that stimulation of chromaffin cells resulted in sustained rises in free calcium, aequorin measurements showed that 55 mM K+ ... More
Coelenterate photoproteins as indicators of cytoplasmic free Ca2+ in small cells.
AuthorsCampbell AK, Dormer RL, Hallett MB
JournalCell Calcium
PubMed ID2861904
The Ca2+-activated photoproteins aequorin and obelin are capable of detecting rapid changes in free Ca2+ over the range 10nM-100uM. Whilst they have been used to quantify free Ca transients in giant cells for some time, their use in small mammalian cells has been restricted because of the difficulty of incorporating ... More
Free calcium increases explosively in activating medaka eggs.
AuthorsRidgway EB, Gilkey JC, Jaffe LF
JournalProc Natl Acad Sci U S A
PubMed ID322135
We have used the calcium-specific light-emitting protein aequorin to follow changes in free calcium concentration during fertilization and cleavage of eggs from medaka, a fresh-water fish. Aequorin-injected medaka eggs show a very low resting glow before they are fertilized, indicating a low calcium concentration in the resting state. Upon activation ... 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
Bioluminescence hybridization assays using recombinant aequorin. Application to the detection of prostate-specific antigen mRNA.
AuthorsGalvan B, Christopoulos TK
JournalAnal Chem
PubMed ID8865762
We developed microtiter well-based bioluminescence hybridization assays using the photoprotein aequorin as a reporter molecule. The target DNA was hybridized simultaneously with a capture probe and a detection probe. The capture probe was immobilized on the wells through digoxigenin/anti-digoxigenin interaction. The detection probe was biotinylated. The hybrids were determined by ... More
Aequorin measurements of free calcium in single heart cells.
AuthorsCobbold PH, Bourne PK
JournalNature
PubMed ID6504155
The performance of the heart depends on the concentrations of free calcium ions in the cytoplasm of the myocytes. However, direct evidence for changes in free Ca concentration in physiological events during response to drugs and in pathogenesis has been difficult to obtain because of technical problems in measuring free ... More
Temporal and spatial dynamics of the periodic increase in intracellular free calcium at fertilization of golden hamster eggs.
AuthorsMiyazaki S, Hashimoto N, Yoshimoto Y, Kishimoto T, Igusa Y, Hiramoto Y
JournalDev Biol
PubMed ID3770302
A series of periodic increases in intracellular free calcium concentration ([Ca2+]i) occurred upon fertilization in golden hamster eggs. The spatial distribution of the Ca2+ transients was investigated in single zona-free, aequorin-injected eggs, inseminated by single sperm. A supersensitive TV camera system for recording Ca2+-aequorin luminescence enabled us to observe the ... More
Vacuole membrane fusion: V0 functions after trans-SNARE pairing and is coupled to the Ca2+-releasing channel.
AuthorsBayer MJ, Reese C, Buhler S, Peters C, Mayer A
JournalJ Cell Biol
PubMed ID12876274
Pore models of membrane fusion postulate that cylinders of integral membrane proteins can initiate a fusion pore after conformational rearrangement of pore subunits. In the fusion of yeast vacuoles, V-ATPase V0 sectors, which contain a central cylinder of membrane integral proteolipid subunits, associate to form a transcomplex that might resemble ... More
The crystal structure of the photoprotein aequorin at 2.3 A resolution.
AuthorsHead JF, Inouye S, Teranishi K, Shimomura O
JournalNature
PubMed ID10830969
Aequorin is a calcium-sensitive photoprotein originally obtained from the jellyfish Aequorea aequorea. Because it has a high sensitivity to calcium ions and is biologically harmless, aequorin is widely used as a probe to monitor intracellular levels of free calcium. The aequorin molecule contains four helix-loop-helix 'EF-hand' domains, of which three ... More
Intracellular ionized calcium changes in squid giant axons monitored by Fura-2 and aequorin.
AuthorsRequena J, Whittembury J, Scarpa A, Brinley JF, Mullins LJ
JournalAnn N Y Acad Sci
PubMed ID1785835
Squid giant axons were injected simultaneously with Ca indicators Fura-2 and aequorin. Fura-2 was calibrated in situ by measuring fluorescence at 510 nm upon UV excitation at 340 nm, 360 nm, and 380 nm with a time-sharing multiple wavelength spectrofluorimeter. Limiting values for dye fluorescence were obtained by allowing a ... More
Slow calcium waves accompany cytokinesis in medaka fish eggs.
AuthorsFluck RA, Miller AL, Jaffe LF
JournalJ Cell Biol
PubMed ID1955473
Animal cells are cleaved by the formation and contraction of an extremely thin actomyosin band. In most cases this contractile band seems to form synchronously around the whole equator of the cleaving cell; however in giant cells it first forms near the mitotic apparatus and then slowly grows outwards over ... More
Measurement of cytoplasmic free Ca2+ concentration in cultured muscle cells by aequorin and quin 2.
AuthorsJames-Kracke MR
JournalAm J Physiol
PubMed ID3094376
Ca2+ has been proposed to regulate expression of the gene for the Ca2+ pump of the sarcoplasmic reticulum in developing chicken myoblasts (A. N. Martonosi, L. Dux, R. L. Terjung, and D. Roufa. Ann. NY Acad. Sci. 402: 485-514, 1982. In the present study, intracellular free Ca2+ ([Ca2+]i) was measured ... More
A solid-phase assay for beta-1,4-galactosyltransferase activity in human serum using recombinant aequorin.
AuthorsZatta PF, Nyame K, Cormier MJ, Mattox SA, Prieto PA, Smith DF, Cummings RD
JournalAnal Biochem
PubMed ID1907813
We have developed a sensitive and rapid solid-phase assay for the serum enzyme UDPGal:beta-D-GlcNAc beta-1,4-galactosyltransferase (beta 1,4-GT) (EC 2.4.1.38) that employs the recombinant bioluminescent protein aequorin as the enzyme label for product detection. The substrate for beta 1,4-GT is a neoglycoprotein, bovine serum albumin containing covalently attached GlcNAc residues (GlcNAc-BSA), ... More
Use of recombinant biotinylated aequorin in microtiter and membrane-based assays: purification of recombinant apoaequorin from Escherichia coli.
Aequorin is a calcium-dependent bioluminescent protein isolated from the hydromedusan Aequorea victoria. The gene for aequorin has been cloned and overexpressed in Escherichia coli [Prasher et al. (1985) Biochem. Biophys. Res. Commun. 126, 1259; Prasher et al. (1987) Biochemistry 26, 1326]. Higher levels of expression have recently been obtained by ... More
Stimulated Ca2+ influx raises mitochondrial free Ca2+ to supramicromolar levels in a pancreatic beta-cell line. Possible role in glucose and agonist-induced insulin secretion.
AuthorsRutter GA, Theler JM, Murgia M, Wollheim CB, Pozzan T, Rizzuto R
JournalJ Biol Chem
PubMed ID8226749
The effects of stimulated Ca2+ influx on cytosolic ([Ca2+]c) or intramitochondrial free Ca2+ ([Ca2+]m) were examined in the new pancreatic beta-cell line, INS-1. [Ca2+]c was monitored by video imaging of single fura-2-loaded INS-1 cells, or in populations of cells transfected with non-targeted (cytosolic) aequorin. [Ca2+]m was measured after transfection with ... More
Source and sinks for the calcium released during fertilization of single sea urchin eggs.
AuthorsEisen A, Reynolds GT
JournalJ Cell Biol
PubMed ID3988799
The source and sinks for the intracellular calcium released during fertilization were examined in single eggs from the sea urchin, Arbacia punctulata. Single eggs were microinjected with the calcium photoprotein, aequorin. The calcium-aequorin luminescence was measured with a microscope-photomultiplier or observed with a microscope-image intensifier-video system. In the normal egg ... More
Rapid ionic modifications during the aequorin-detected calcium transient in a skinned canine cardiac Purkinje cell.
AuthorsFabiato A
JournalJ Gen Physiol
PubMed ID3981128
A microprocessor-controlled system of microinjections and microaspirations has been developed to change, within approximately 1 ms, the [free Ca2+] at the outer surface of the sarcoplasmic reticulum (SR) wrapped around individual myofibrils (0.3-0.4 micron radius) of a skinned canine cardiac Purkinje cell (2.5-4.5 micron overall radius) at different phases of ... More
Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). A critical evaluation.
AuthorsBrini M, Marsault R, Bastianutto C, Alvarez J, Pozzan T, Rizzuto R
JournalJ Biol Chem
PubMed ID7730373
Targeted recombinant aequorins represent to date the most specific means of monitoring [Ca2+] in subcellular organelles (Rizzuto, R., Simpson, A. W. M., Brini, M., and Pozzan, T. (1992) Nature 358, 325-328; Brini, M., Murgia, M., Pasti, L., Picard, D., Pozzan, T., and Rizzuto, R. (1993) EMBO J. 12, 4813-4819; Kendall, ... More