Bioluminescent immunoassay for serine/threonine protein kinase activity using an aequorin-labeled monoclonal antibody and a synthetic peptide as a substrate.

A bioluminescent immunoassay system was developed to determine serine/threonine protein kinase activity using an aequorin-labeled monoclonal antibody and a synthetic peptide as the substrate. A monoclonal antibody against the synthetic phosphorylated serine peptide (K9P peptide) of histone H3 (19 amino acid residues), referred to as the H3S10P antibody, was chemically conjugated to maleimide-activated aequorin to prepare aequorin-labeled H3S10P (AQ-S-H3S10P). For the serine/threonine kinase assay, a non-phosphorylated serine peptide (K9C peptide) coated on a microplate was incubated with serine/threonine protein kinase in the presence of ATP and Mg2+. The resulting phosphorylated K9C peptides (K9P peptide) were identified using AQ-S-H3S10P. Thus, after the removal of unbound AQ-S-H3S10P though washing, the serine/threonine kinase activity was determined by the luminescence activity of aequorin from AQ-S-H3S10P bound to the K9P peptide. This assay system, in combination with the K9C peptide and AQ-S-H3S10P, could be used to screen inhibitors of various serine/threonine protein kinases in general.

Bioluminescence Detection of Superoxide Anion Using Aequorin.

Although the superoxide anion (O2-·) is generated during normal cellular respiration and has fundamental roles in a wide range of cellular processes, such as cell proliferation, migration, apoptosis, and homeostasis, its dysregulation is associated with a variety of diseases. Regarding these prominent roles in biological systems, the development of accurate methods for quantification of superoxide anion has attracted tremendous research attention. Here, we evaluated aequorin, a calcium-dependent photoprotein, as a potential bioluminescent reporter protein of superoxide anion. The mechanism is based on the measurement of aequorin bioluminescence, where the lower the concentration of coelenterazine under the oxidation of superoxide anion, the lower the amount aequorin regeneration, leading to a decrease in bioluminescence. The bioluminescence intensity of aequorin was proportional to the concentration of superoxide anion in the range from 4 to 40 000 pM with a detection limit (S/N = 3) of 1.2 pM, which was 5000-fold lower than those of the chemiluminescence methods. The proposed method exhibited high sensitivity and has been successfully applied to the determination of superoxide anion in the plant cell samples. The results could suggest a photoprotein-based bioluminescence system as a highly sensitive, specific, and simple bioluminescent probe for in vitro detection of superoxide anion.

Enhanced elevations of hypo-osmotic shock-induced cytosolic and nucleic calcium concentrations in tobacco cells by pretreatment with dimethyl sulfoxide.

Dimethyl sulfoxide (DMSO) is a dipolar aprotic solvent widely used in biological assays. Here, we observed that DMSO enhanced the hypo-osmotically induced increases in the concentration of Ca2+ in cytosolic and nucleic compartments in the transgenic cell-lines of tobacco (BY-2) expressing aequorin.

Methanol induces cytosolic calcium variations, membrane depolarization and ethylene production in arabidopsis and tobacco.

Background and Aims: Methanol is a volatile organic compound released from plants through the action of pectin methylesterases (PMEs), which demethylesterify cell wall pectins. Plant PMEs play a role in developmental processes but also in responses to herbivory and infection by fungal or bacterial pathogens. However, molecular mechanisms that explain how methanol could affect plant defences remain poorly understood. Methods: Using cultured cells and seedlings from Arabidopsis thaliana and tobacco BY2 expressing the apoaequorin gene, allowing quantification of cytosolic Ca2+, a reactive oxygen species (ROS) probe (CLA, Cypridina luciferin analogue) and electrophysiological techniques, we followed early plant cell responses to exogenously supplied methanol applied as a liquid or as volatile. Key Results: Methanol induces cytosolic Ca2+ variations that involve Ca2+ influx through the plasma membrane and Ca2+ release from internal stores. Our data further suggest that these Ca2+ variations could interact with different ROS and support a signalling pathway leading to well known plant responses to pathogens such as plasma membrane depolarization through anion channel regulation and ethylene synthesis. Conclusions: Methanol is not only a by-product of PME activities, and our data suggest that [Ca2+]cyt variations could participate in signalling processes induced by methanol upstream of plant defence responses.

Bioluminescent and biochemical properties of Cys-free Ca2+-regulated photoproteins obelin and aequorin.

Bioluminescence of a variety of marine coelenterates is determined by Ca2+-regulated photoproteins. A strong interest in these proteins is for their wide analytical potential as intracellular calcium indicators and labels for in vitro binding assays. The presently known hydromedusan Ca2+-regulated photoproteins contain three (aequorin and clytin) or five (obelin and mitrocomin) cysteine residues with one of them strictly conserved. We have constructed Cys-free aequorin and obelin by substitution of all cysteines to serine residues. Such mutants should be of interest for researchers by the possibility to avoid the incubation with dithiothreitol (or ß-mercaptoethanol) required for producing an active photoprotein that is important for some prospective analytical assays in which the photoprotein is genetically fused with a target protein sensitive to the reducing agents. Cys-free mutants were expressed in Escherichia coli, purified, and characterized regarding the efficiency of photoprotein complex formation, functional activity, and conformational stability. The replacement of cysteine residues has been demonstrated to affect different properties of aequorin and obelin. Cys-free aequorin displays a two-fold lower specific bioluminescence activity but preserves similar activation properties and light emission kinetics compared to the wild-type aequorin. In contrast, Cys-free obelin retains only ~10% of the bioluminescence activity of wild-type obelin as well as binding coelenterazine and forming active photoprotein much less effectively. In addition, the substitution of Cys residues drastically changes the bioluminescence kinetics of obelin completely eliminating a "fast" component from the light signal decay curve. At the same time, the replacement of Cys residues increases conformational flexibility of both aequorin and obelin molecules, but again, the effect is more prominent in the case of obelin. The values of thermal midpoints of unfolding (Tm) were determined to be 53.3±0.2 and 44.6±0.4°C for aequorin and Cys-free aequorin, and 49.1±0.1 and 28.8±0.3°C for obelin and Cys-free obelin, respectively. Thus, so far only Cys-free aequorin is suitable as a partner for fusing with a tag sensitive to reducing agents since the aequorin mutant preserves almost 50% of the bioluminescent activity and can be produced with a substantial yield.

Red-Shifted Aequorin Variants Incorporating Non-Canonical Amino Acids: Applications in In Vivo Imaging.

The increased importance of in vivo diagnostics has posed new demands for imaging technologies. In that regard, there is a need for imaging molecules capable of expanding the applications of current state-of-the-art imaging in vivo diagnostics. To that end, there is a desire for new reporter molecules capable of providing strong signals, are non-toxic, and can be tailored to diagnose or monitor the progression of a number of diseases. Aequorin is a non-toxic photoprotein that can be used as a sensitive marker for bioluminescence in vivo imaging. The sensitivity of aequorin is due to the fact that bioluminescence is a rare phenomenon in nature and, therefore, it does not suffer from autofluorescence, which contributes to background emission. Emission of bioluminescence in the blue-region of the spectrum by aequorin only occurs when calcium, and its luciferin coelenterazine, are bound to the protein and trigger a biochemical reaction that results in light generation. It is this reaction that endows aequorin with unique characteristics, making it ideally suited for a number of applications in bioanalysis and imaging. Herein we report the site-specific incorporation of non-canonical or non-natural amino acids and several coelenterazine analogues, resulting in a catalog of 72 cysteine-free, aequorin variants which expand the potential applications of these photoproteins by providing several red-shifted mutants better suited to use in vivo. In vivo studies in mouse models using the transparent tissue of the eye confirmed the activity of the aequorin variants incorporating L-4-iodophehylalanine and L-4-methoxyphenylalanine after injection into the eye and topical addition of coelenterazine. The signal also remained localized within the eye. This is the first time that aequorin variants incorporating non-canonical amino acids have shown to be active in vivo and useful as reporters in bioluminescence imaging.

Expression and reconstitution of the bioluminescent Ca(2+) reporter aequorin in human embryonic stem cells, and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes.

In order to develop a novel method of visualizing possible Ca(2+) signaling during the early differentiation of hESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca(2+) reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca(2+) transients (generated by release from intracellular stores) were detected in 1-12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or CaCl2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor (IP3R) agonist, small Ca(2+) transients were generated from day 1 onward. That ATP was inducing Ca(2+) release from functional IP3Rs was demonstrated by treatment with 2-APB, a known IP3R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor (RyR) agonist, a minimal Ca(2+) response was observed at day 8 of differentiation only. Thus, our data indicate that unlike RyRs, IP3Rs are present and continually functional at these early stages of cardiomyocyte differentiation.

Aequorin as Intracellular Ca2+ Indicator Incorporated in Follicular Lymphoma Cells by Hypoosmotic Shock Treatment.

Natural proteins can be used in measuring intracellular Ca(2+) concentration. As one of the Ca(2+)- regulated photoproteins, aequorin has several advantages in comparison to widely used Ca(2+) fluorescence indicators (e.g., fura-2, indo-1 and fluo-3), including high dynamic range and resistance to motion artefacts. However, incorporation of aequorin into cells remains a challenge. Hypoosmotic shock treatment was optimized and used as a method for loading aequorin into the cytoplasm of follicular lymphoma cells. Measurement of aequorin luminescence in the cells was performed using a luminometer with a sensitive photomultiplier tube and the luminescence intensity was recalculated into intracellular [Ca(2+)]. The value of (0.85 ± 0.52)·10-6 M was found. We show that the optimized method of incorporation was effective for loading aequorin into follicular lymphoma cells in vitro. The cell viability remains high immediately after the procedure. This method can also be used for measuring intracellular Ca(2+) concentration in other types of non-adherent cells.

Ca(2+) homeostasis in the budding yeast Saccharomyces cerevisiae: Impact of ER/Golgi Ca(2+) storage.

Yeast has proven to be a powerful tool to elucidate the molecular aspects of several biological processes in higher eukaryotes. As in mammalian cells, yeast intracellular Ca(2+) signalling is crucial for a myriad of biological processes. Yeast cells also bear homologs of the major components of the Ca(2+) signalling toolkit in mammalian cells, including channels, co-transporters and pumps. Using yeast single- and multiple-gene deletion strains of various plasma membrane and organellar Ca(2+) transporters, combined with manipulations to estimate intracellular Ca(2+) storage, we evaluated the contribution of individual transport systems to intracellular Ca(2+) homeostasis. Yeast strains lacking Pmr1 and/or Cod1, two ion pumps implicated in ER/Golgi Ca(2+) homeostasis, displayed a fragmented vacuolar phenotype and showed increased vacuolar Ca(2+) uptake and Ca(2+) influx across the plasma membrane. In the pmr1Δ strain, these effects were insensitive to calcineurin activity, independent of Cch1/Mid1 Ca(2+) channels and Pmc1 but required Vcx1. By contrast, in the cod1Δ strain increased vacuolar Ca(2+) uptake was not affected by Vcx1 deletion but was largely dependent on Pmc1 activity. Our analysis further corroborates the distinct roles of Vcx1 and Pmc1 in vacuolar Ca(2+) uptake and point to the existence of not-yet identified Ca(2+) influx pathways.

p53 at the endoplasmic reticulum regulates apoptosis in a Ca2+-dependent manner.

The tumor suppressor p53 is a key protein in preventing cell transformation and tumor progression. Activated by a variety of stimuli, p53 regulates cell-cycle arrest and apoptosis. Along with its well-documented transcriptional control over cell-death programs within the nucleus, p53 exerts crucial although still poorly understood functions in the cytoplasm, directly modulating the apoptotic response at the mitochondrial level. Calcium (Ca(2+)) transfer between the endoplasmic reticulum (ER) and mitochondria represents a critical signal in the induction of apoptosis. However, the mechanism controlling this flux in response to stress stimuli remains largely unknown. Here we show that, in the cytoplasm, WT p53 localizes at the ER and at specialized contact domains between the ER and mitochondria (mitochondria-associated membranes). We demonstrate that, upon stress stimuli, WT p53 accumulates at these sites and modulates Ca(2+) homeostasis. Mechanistically, upon activation, WT p53 directly binds to the sarco/ER Ca(2+)-ATPase (SERCA) pump at the ER, changing its oxidative state and thus leading to an increased Ca(2+) load, followed by an enhanced transfer to mitochondria. The consequent mitochondrial Ca(2+) overload causes in turn alterations in the morphology of this organelle and induction of apoptosis. Pharmacological inactivation of WT p53 or naturally occurring p53 missense mutants inhibits SERCA pump activity at the ER, leading to a reduction of the Ca(2+) signaling from the ER to mitochondria. These findings define a critical nonnuclear function of p53 in regulating Ca(2+) signal-dependent apoptosis.

Differential calcium handling by the cis and trans regions of the Golgi apparatus.

High Ca2+ content in the Golgi apparatus (Go) is essential for protein processing and sorting. In addition, the Go can shape the cytosolic Ca2+ signals by releasing or sequestering Ca2+. We generated two new aequorin-based Ca2+ probes to specifically measure Ca2+ in the cis/cis-to-medial-Go (cGo) or the trans-Go (tGo). Ca2+ homoeostasis in these compartments and in the endoplasmic reticulum (ER) has been studied and compared. Moreover, the relative size of each subcompartment was estimated from aequorin consumption. We found that the cGo accumulates Ca2+ to high concentrations (150-300 µM) through the sarco plasmic/endoplasmic reticulum Ca2+-ATPase (SERCA). The tGo, in turn, is divided into two subcompartments: tGo1 and tGo2. The subcompartment tGo1 contains 20% of the aequorin and has a high internal [Ca2+]; Ca2+ is accumulated in this subcompartment via the secretory pathway Ca2+-ATPase 1 (SPCA-1) at a very high affinity (K50=30 nM). The subcompartment tGo2 contains 80% of aequorin, has a lower [Ca2+] and no SPCA-1 activity; Ca2+ uptake happens through SERCA and is slower than in tGo1. The two tGo subcompartments, tGo1 and tGo2, are diffusionally isolated. Inositol trisphosphate mobilizes Ca2+ from the cGo and tGo2, but not from tGo1, whereas caffeine releases Ca2+ from all the Golgi regions, and nicotinic acid dinucleotide phosphate and cADP ribose from none.

Compartment calcium model of frog skeletal muscle during activation.

Skeletal muscle contraction is triggered by a rise in calcium (Ca(2+)) concentration in the myofibrillar space. The objective of this study was to develop a voltage dependent compartment model of Ca(2+) dynamics in frog skeletal muscle fibers. The compartment model corresponds to the myofibrillar space (MS) and a calcium store, the sarcoplasmic reticulum (SR). Ca(2+) is released from the SR to the MS based on the voltage and is able to bind to several proteins in the MS. We use a detailed model to account for voltage dependent Ca(2+) release and inactivation. With this model, we are able to match previous experimental data for Ca(2+) release and binding to proteins for an applied (fixed) voltage. We explore the sensitivity of parameters in the model and illustrate the importance of inactivation of the SR; during a long depolarization, the SR must be inactivated in order to achieve realistic Ca(2+) concentrations in the MS. A Hodgkin Huxley type model was also developed to describe voltage at the surface membrane using electrophysiological data from previous experiments. This voltage model was then used as the time dependent voltage to determine Ca(2+) release from the SR. With this fully coupled model, we were able to match previous experimental results for Ca(2+) concentrations for a given applied current. Additionally, we examined simulated Ca(2+) concentrations in the case of twitch and tetanus, corresponding to different applied currents. The developed model is robust and reproduces many aspects of voltage dependent calcium signaling in frog skeletal muscle fibers. This modeling framework provides a platform for future studies of excitation contraction coupling in skeletal muscle fibers.

Methods to measure cytoplasmic and mitochondrial Ca(2+) concentration using Ca(2+)-sensitive dyes.

Ca(2+) is a ubiquitous second messenger that is involved in regulation of various signaling pathways. Cytoplasmic Ca(2+) is maintained at low concentrations (~100 nM) by many active mechanisms. Increases in intracellular Ca(2+) concentration ([Ca(2+)]i) indeed can initiate multiple signaling pathways, depending both on their pattern and subcellular localization. In T cells, the stimulation of T-cell receptor leads to an increase in [Ca(2+)]i upon the opening of Ca(2+) release-activated calcium (CRAC) channels. T cells can actually sustain high [Ca(2+)]i for several hours, resulting in the activation of transcriptional programs orchestrated by members of the nuclear factor of activated T-cell (NFAT) protein family. Here, we describe an imaging method widely employed to measure cytoplasmic [Ca(2+)] in naïve and effector T cells based on the ratiometric dye Fura-2. Furthermore, we discuss a pharmacological method relying on an inhibitor of CRAC channels, 2-aminoethyldiphenyl borate, to validate the role of CRAC channels in cytoplasmic Ca(2+) elevation. Finally, we describe an approach to measure mitochondrial [Ca(2+)] based on another fluorescent dye, Rhod-2. With appropriate variations, our methodological approach can be employed to assess the effect and regulation of cytosolic and mitochondrial Ca(2+) waves in multiple experimental settings, including cultured cancer cells.

Methods to measure intracellular Ca(2+) fluxes with organelle-targeted aequorin-based probes.

The photoprotein aequorin generates blue light upon binding of Ca(2+) ions. Together with its very low Ca(2+)-buffering capacity and the possibility to add specific targeting sequences, this property has rendered aequorin particularly suitable to monitor Ca(2+) concentrations in specific subcellular compartments. Recently, a new generation of genetically encoded Ca(2+) probes has been developed by fusing Ca(2+)-responsive elements with the green fluorescent protein (GFP). Aequorin has also been employed to this aim, resulting in an aequorin-GFP chimera with the Ca(2+) sensitivity of aequorin and the fluorescent properties of GFP. This setup has actually solved the major limitation of aequorin, for example, its poor ability to emit light, which rendered it inappropriate for the monitoring of Ca(2+) waves at the single-cell level by imaging. In spite of the numerous genetically encoded Ca(2+) indicators that are currently available, aequorin-based probes remain the method of election when an accurate quantification of Ca(2+) levels is required. Here, we describe currently available aequorin variants and their use for monitoring Ca(2+) waves in specific subcellular compartments. Among various applications, this method is relevant for the study of the alterations of Ca(2+) homeostasis that accompany oncogenesis, tumor progression, and response to therapy.

The intracellular delivery of TAT-aequorin reveals calcium-mediated sensing of environmental and symbiotic signals by the arbuscular mycorrhizal fungus Gigaspora margarita.

Arbuscular mycorrhiza (AM) is an ecologically relevant symbiosis between most land plants and Glomeromycota fungi. The peculiar traits of AM fungi have so far limited traditional approaches such as genetic transformation. The aim of this work was to investigate whether the protein transduction domain of the HIV-1 transactivator of transcription (TAT) protein, previously shown to act as a potent nanocarrier for macromolecule delivery in both animal and plant cells, may translocate protein cargoes into AM fungi. We evaluated the internalization into germinated spores of Gigaspora margarita of two recombinant TAT fusion proteins consisting of either a fluorescent (GFP) or a luminescent (aequorin) reporter linked to the TAT peptide. Both TAT-fused proteins were found to enter AM fungal mycelia after a short incubation period (5-10 min). Ca2+ measurements in G. margarita mycelia pre-incubated with TAT-aequorin demonstrated the occurrence of changes in the intracellular free Ca2+ concentration in response to relevant stimuli, such as touch, cold, salinity, and strigolactones, symbiosis-related plant signals. These data indicate that the cell-penetrating properties of the TAT peptide can be used as an effective strategy for intracellularly delivering proteins of interest and shed new light on Ca2+ homeostasis and signalling in AM fungi.

Protection of tobacco cells from oxidative copper toxicity by catalytically active metal-binding DNA oligomers.

The impact of copper ions on the oxidative and calcium signal transductions, leading to cell death in plant cells, have been documented. Copper induces a series of biological and chemical reactions in plant cells including the oxidative burst reflecting the production of reactive oxygen species and the stimulation of calcium channel opening allowing a transient increase in cytosolic calcium concentrations. These early events, completed within a few minutes after the contact with copper, are known to trigger the development of cell death. The effects of DNA fragments with copper-binding motifs as novel plant cell-protecting agents were assessed using cell suspension cultures of transgenic tobacco (Nicotiana tabacum L., cell line BY-2) expressing the aequorin gene. The addition of GC-rich double-stranded DNA fragments, prior to the addition of copper ions, effectively blocked both the copper-induced calcium influx and cell death. In addition, the DNA-Cu complex examined was shown to possess superoxide-scavenging catalytic activity, suggesting that DNA-mediated protection of the cells from copper toxicity is due to the removal of superoxide. Lastly, a possible mechanism of DNA-Cu interaction and future applications of these DNA fragments in the protection of plant roots from metal toxicity or in aid of phyto-remediation processes are discussed.

Development of aequorin-based mast cell nanosensor for rapid identification of botulinum neurotoxin type B.

An aequorin-based mast cell sensor was developed for rapid identification and detection of protein toxins. To monitor mast cell activation and to improve the sensitivity of detection, aequorin was stably expressed in the cells and used as an indicator of calcium flux and the stable cell line was created. The procedures for preparing cells were optimized to improve the quantum yield and sensitivity of detection. The cells sensitized with anti-DNP (dinitrophenyl) IgE were capable of detecting as little as 0.1 ng/mL DNP-BSA in less than 2 min. To demonstrate the utility of this mast cell sensor in detecting protein toxins, an IgE chimeric protein (p21-Fcε) was created. This was achieved by fusing the Fc region of IgE antibody to a 21-amino acid peptide (p21) derived from residues 40-60 of synaptotagmin II (Syt II), the protein receptor of botulinum neurotoxin type B (BoNT/B). In addition, magnetic beads linked with anti-BoNT/B polyclonal antibody were prepared to capture BoNT/B molecules in solution to make targets multivalent and concentrated. The p21-Fcε binds to FcsRI receptors on RBL (Rat basophilic leukemia) cells and can be cross-linked by BoNT/B captured by the magnetic beads. This led to cell activation and Ca2+ flux indicated by an increase of quantum yield. This assay can detect as little as 100 pM (15 ng/mL) BoNT/B in less than 1 hr, which included the initial sample preparation time. This study lays a foundation for detecting other protein toxins using mast cell sensors.

Measurements of Ca²âº concentration with recombinant targeted luminescent probes.

In the last two decades the study of Ca(2+) homeostasis in living cells has been enhanced by the explosive development of genetically encoded Ca(2+)-indicators. The cloning of the Ca(2+)-sensitive photoprotein aequorin and of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has been enormously advantageous. As polypeptides, aequorin and GFP allow their endogenous production in cell systems as diverse as bacteria, yeast, slime molds, plants, and mammalian cells. Moreover, it is possible to specifically localize them within the cell by including defined targeting signals in the amino acid sequence. These two proteins have been extensively engineered to obtain several recombinant probes for different biological parameters, among which Ca(2+) concentration reporters are probably the most relevant. The GFP-based Ca(2+) probes and aequorin are widely employed in the study of intracellular Ca(2+) homeostasis. The new generation of bioluminescent probes that couple the Ca(2+) sensitivity of aequorin to GFP fluorescence emission allows real-time measurements of subcellular Ca(2+) changes in single cell imaging experiments and the video-imaging of Ca(2+) concentrations changes in live transgenic animals that express GFP-aequorin bifunctional probes.

In vivo analysis of the calcium signature in the plant Golgi apparatus reveals unique dynamics.

The Golgi apparatus is thought to play a role in calcium homeostasis in plant cells. However, the calcium dynamics in this organelle is unknown in plants. To monitor the [Ca2+]Golgiin vivo, we obtained and analyzed Arabidopsis thaliana plants that express aequorin in the Golgi. Our results show that free [Ca2+] levels in the Golgi are higher than in the cytosol (0.70 µM vs. 0.05 µM, respectively). Stimuli such as cold shock, mechanical stimulation and hyperosmotic stress, led to a transient increase in cytosolic calcium; however, no instant change in the [Ca2+]Golgi concentration was detected. Nevertheless, a delayed increase in the [Ca2+]Golgi up to 2-3 µM was observed. Cyclopiazonic acid and thapsigargin inhibited the stimuli-induced [Ca2+]Golgi increase, suggesting that [Ca2+]Golgi levels are dependent upon the activity of Ca2+-ATPases. Treatment of these plants with the synthetic auxin analog, 2,4-dichlorophenoxy acetic acid (2,4-D), produced a slow decrease of free calcium in the organelle. Our results indicate that the plant Golgi apparatus is not involved in the generation of cytosolic calcium transients and exhibits its own dynamics modulated in part by the activity of Ca2+ pumps and hormones.

Purification of histidine-tagged aequorin with a reactive cysteine residue for chemical conjugations and its application for bioluminescent sandwich immunoassays.

Highly purified histidine-tagged aequorin with a reactive cysteine residue (His-Cys4-aequorin) was obtained from the periplasmic space of Escherichia coli cells by nickel-chelate affinity chromatography and hydrophobic chromatography. The procedure yielded 40.3mg of His-Cys4-aequorin from 2L of cultured cells with over 95% purity. The chemical conjugates of His-Cys4-aequorin with maleimide-activated streptavidin and maleimide-activated biotin were prepared without significant loss of luminescence activity and were applied to the bioluminescent sandwich immunoassay for α-fetoprotein (AFP) as a model analyte. The measurable range of AFP by these conjugates was 0.01-100 ng/ml and the sensitivities were similar to that using aequorin-labeled specific antibody and amino-biotinylated aequorin.