Comparative aspects of the calcium-sensitive photoproteins aequorin and obelin.

1. The calcium-dependency of the process of light emission has been investigated for the photoproteins aequorin and obelin. 2. The experimental curves of light production, expressed as a percentage of the maximal rate of utilisation, versus pCa are accurately predicted by the cooperative action of at least 2Ca-2+ for aequorin and at least 3Ca-2+ for obelin. 3. At low total monovalent cation concentrations, a pH change from 6.8 to 7.1 shifts the light production vs pCa curve by approx. 0.2 pCa units to the right for aequorin, while that for obelin is shifted by some 0.37 pCa units. 4. Other monovalent cations, such as Na+ are able to compete with Ca-2+ for the active sites of aequorin and also shift the light production vs pCa curve to the right. There is no apparent change in the calcium stoichiometry for light production under these conditions. 5. The same calcium stoichiometry for light emission was also obtained for aequorin or obelin in the presence of either unbuffered Ca-2+ solutions or of calcium/EGTA buffers.     

Studies on the luminescent response of the Ca2+-activated photoprotein,obelin

1. The kinetics and stoicheiometry of the Ca²⁺-activated luminescent reaction of the photoprotein obelin were studied at different temperatures and in the presence of various substances, including the physiologically occurring cations K⁺, Na⁺, Ca²⁺, Mg²⁺ and H⁺. 2. The results suggest Ca²⁺-independent rates of rise and fall in obelin luminescence following sudden changes in [Ca²⁺] and indicate that changes in [Ca²⁺] over the range 1 · 10^?6?3 · 10^?4 M are followed significantly faster by the obelin response (approx. 3 ms delay at 20°C) than by the aequorin response (approx. 10 ms delay at 20°C). 3. Obelin was found to emit low-intensity light (less than 10^?6 of the maximum Ca²⁺-activated response), which was independent of Ca²⁺ at concentrations below about 10^?7 M. The level of this Ca²⁺-independent light emission is sensitive to temperature and the ionic composition of the solution. 4. The log-log plot of light intensity against ionized Ca indicates a maximum slope of 2.5, suggesting the involvement of three Ca ions in the luminescent reaction. 5. Increases in the concentration of K⁺, Na⁺, Mg²⁺ and H⁺ generally shift the Ca²⁺ activation curve for obelin toward higher Ca²⁺ concentrations. These cations can also affect the maximum rate of obelin utilization at more extreme concentrations. 6. The maximal rate of obelin utilization was also affected to varying degrees by the presence of uncharged substances such as glucose, sucrose and polyvinylpyrrolidone. However, neither the sensitivity of obelin to Ca²⁺ nor the quantum yield were modified by these substances. 7. Caffeine (less than 20 mM), procaine (less than 20 mM) and sodium dantrolene (saturated solution), substances known to modify cellular Ca²⁺ movements, had little effect on the Ca²⁺-induced luminescent reaction. The general anaesthetics chlorpromazine and halothane appeared to lower greatly the quantum yield without, however, modifying the maximum rate of obelin utilization. 8. A scheme of reaction for obelin activation by Ca²⁺ is presented which adequately explains the experimental observations and allows one to make accurate predictions regarding the relative obelin respones under a variety of ionic conditions at room temperature.     

Rapid Kinetic Studies of the Light Emitting Protein Aequorin

SHIMUMURA et al.^1,2 isolated from luminescent jelly fish (Aequora forskalea) a protein (aequorin) which on addition of Ca²⁺ emits light. In contrast to other bioluminescent compounds, aequorin luminescence requires neither oxygen nor FMNH₂, ATP nor long chain aldehydes, but only Ca²⁺. Recently, other bioluminescent proteins reacting only with Ca²⁺ have been isolated and termed Ca²⁺-activated photoproteins^3,4. Since the intensity of the emitted light varies with the Ca²⁺ concentration, aequorin and related proteins can serve as useful tools for detecting small changes in Ca²⁺ concentration in biological systems^5,6. Little is known, however, about the mechanism of this reaction. Since light emission from aequorin occurs within a few milliseconds of rapid mixing with Ca²⁺, kinetic studies are possible only with rapid mixing instruments, stopped and continuous flow. Hastings et al. studied the kinetics of aequorin using stopped flow and double stopped flow apparatuses⁷. Because both have a dead time of about 2 ms, which is in the same range as the rise time of the light emission from aequorin, they were unable to establish a Ca²⁺ dependence for the rate of rise of the light emission. Ca²⁺ dependence of this rate might be very important for delineation of the reaction mechanism.     

Free calcium changes associated with hormone action in amphibian oocytes

Ca²⁺-sensitive electrodes and the photoproteins obelin and aequorin were used with the oocytes of the anuran Xenopus laevis and the urodeles Ambystoma mexicanum and Pleurodeles waltlii in order to detect any changes in internal free Ca²⁺ which might result from progesterone or agonist stimulation. A dramatic Ca²⁺ surge was recorded: from 0.7 × 10^?6M in the unstimulated oocyte to 7 × 10^?6M after stimulation but before germinal vesicle breakdown (GVBD). Ca²⁺ efflux was also measured, but it accounted for less than 0.2% of the internal Ca²⁺ transient; this efflux did not take place in the absence of external calcium. The Ca²⁺ surge and maturation in response to progesterone, p-hydroxymethylenesulfonate (PHMPS), or Mn²⁺ occurred normally even when divalent cations were absent from the external medium. In contrast, external divalent cations were necessary for the induction of meiosis and a Ca²⁺ transient by the K⁺ ionophore valinomycin. HCO₃^? also triggers meiosis and causes Ca²⁺ release, but the release occurs with quite different kinetics. Incompletely grown or seasonally dormant oocytes as well as 10 mM theophilline- or procaine-treated oocytes neither release Ca²⁺ nor respond to the hormone. We conclude that intracellular released Ca²⁺ is likely to be the major “second messenger” following hormone stimulation in amphibian oocytes as in starfish.     

ATP-dependent calcium transport in membrane vesicles of the cyanobacterium, Anabaena variabilis

Transport of Ca²⁺ in membrane vesicles of the cyanobacterium Anabaena variabilis has been investigated. The light membranes previously shown to carry a Mg²⁺-dependent, Ca²⁺-stimulated ATPase (Lockau, W. and Pfeffer, S. (1982) Z. Naturforsch. 37C, 658–664) accumulate Ca²⁺ upon addition of ATP, whereas the (heavier) thylakoids do not. A stoichiometry of 0.3 Ca²⁺ taken up per ATP hydrolyzed has been determined from initial rates, which is considered to be an underestimation of the true stoichiometry of the pump. Calcium transport and Ca²⁺-stimulated ATPase activity are both sensitive to Na₃VO₄ (an inhibitor of ATPases forming a phosphorylated intermediate), show the same pH optimum and a comparable dependence on ATP concentration. Calcium transport is also supported by nucleoside triphosphates other than ATP, although at lower rates. Accumulation of calcium is abolished by an ionophore of divalent cations, ionophore A23187, but is resistant to ionophores of monovalent cations and to the inhibitor of F₁-F₀-type ATPases, N,N′-dicyclohexylcarbodiimide. It is concluded that the ATPase is a primary calcium pump.     

Luminescence of Ca2+-activated photoprotein obelin initiated by NaOCl and MnCl2

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 NaOCI to the mixture causes singlet oxygen formation (stimulated by Mn²⁺ ions), and singlet oxygen initiates the light-emitting reaction.     

A Comparison between Quin-2 and Aequorin as Indicators of Cytoplasmic Calcium Levels in Higher Plant Cell Protoplasts

Assessment of the regulation of plant metabolism by the calcium ion requires a knowledge of its intracellular levels and dynamics. Technical problems have prevented direct measurement of the concentration of intracellular Ca²⁺ in plant cells in all but a few cases. In this study we show that electropermeabilized protoplasts of Daucus carota and Hordeum vulgare took up the Ca²⁺ indicating fluorescent dye methoxyquinoline(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (Quin-2) and the Ca²⁺ indicating photoprotein, aequorin. These protoplasts subsequently recovered their plasma membrane integrity. However, up to 10% of intracellularly trapped Quin-2 was associated with a protoplast vacuolar fraction. Also, Quin-2 loading reduced total ATP levels by approximately 60% and inhibited subsequent protoplast division whereas aequorin loading reduced ATP content by only 20% and did not prevent division. Therefore, the basal cytoplasmic Ca²⁺ level measured with aequorin (less than 200 nanomolar) may more reliably reflect that found in vivo in the unperturbed protoplast than that measured with Quin-2 (120-360 nanomolar). However, measurements made with aequorin were found to be inaccurate at Ca²⁺ levels below 200 nanomolar, Quin-2 proving complementary in indicating these low Ca²⁺ concentrations. Cytosolic Ca²⁺ was observed to increase on treatment with azide and silver ions.     

Calcium homeostasis in Pseudomonas aeruginosa requires multiple transporters and modulates swarming motility

Pseudomonas aeruginosa is an opportunistic human pathogen causing severe acute and chronic infections. Earlier we have shown that calcium (Ca²⁺) induces P. aeruginosa biofilm formation and production of virulence factors. To enable further studies of the regulatory role of Ca²⁺, we characterized Ca²⁺ homeostasis in P. aeruginosa PAO1 cells. By using Ca²⁺-binding photoprotein aequorin, we determined that the concentration of free intracellular Ca²⁺ ([Ca²⁺]_in) is 0.14 ± 0.05 μM. In response to external Ca²⁺, the [Ca²⁺]_in quickly increased at least 13-fold followed by a multi-phase decline by up to 73%. Growth at elevated Ca²⁺ modulated this response. Treatment with inhibitors known to affect Ca²⁺ channels, monovalent cations gradient, or P-type and F-type ATPases impaired [Ca²⁺]_in response, suggesting the importance of the corresponding mechanisms in Ca²⁺ homeostasis. To identify Ca²⁺ transporters maintaining this homeostasis, bioinformatic and LC–MS/MS-based membrane proteomic analyses were used. [Ca²⁺]_in homeostasis was monitored for seven Ca²⁺-affected and eleven bioinformatically predicted transporters by using transposon insertion mutants. Disruption of P-type ATPases PA2435, PA3920, and ion exchanger PA2092 significantly impaired Ca²⁺ homeostasis. The lack of PA3920 and vanadate treatment abolished Ca²⁺-induced swarming, suggesting the role of the P-type ATPase in regulating P. aeruginosa response to Ca²⁺.     

Radial spread of aequorin Ca2+ signal in single frog skeletal muscle fibers

The Ca²⁺-sensitive photoprotein aequorin was injected into single frog skeletal muscle fibers, and the intracellular aequorin light intensity during muscle activation with different maneuvers was mapped with digital imaging microscopy. During 50 Hz electrical activation (tetanus), the aequorin light intensity from different locations in the muscle fiber rose with very similar time course. Caffeine (10 mM) application, on the other hand, caused aequorin light signals to show significantly different time courses, with an earlier increase in Ca²⁺ concentration near the surface of the fiber than near the core. The non-uniform rise of intracellular Ca²⁺ concentration with caffeine treatment is consistent with the slow inward diffusion of caffeine and subsequent Ca²⁺ release from sarcoplasmic reticulum.     

Focal increases in [Ca]i may account for apparent low Ca sensitivity in swine carotid artery

Estimates of [Ca²⁺]_i sensitivity in intact smooth muscle are frequently obtained by measuring [Ca²⁺]_i with indicators such as aequorin or Fura-2. We investigated whether focal in increases in [Ca²⁺]_i could impair such measures of [Ca²⁺]_i sensitivity. Stimulation of swine carotid artery with 10 μM histamine increased aequorin estimated [Ca²⁺]_i, Fura-2 estimated [Ca²⁺]_i and Ca²⁺ sensitivity without significantly altering the aequorin/Fura-2 ratio (an estimate of [Ca²⁺]_i homogeneity). Subsequent inhibition of Na⁺/Ca²⁺ exchange by replacement of Na⁺ in the PSS with choline⁺ significantly increased aequorin-estimated [Ca²⁺]_i but only minimally increased Fura-2 estimated [Ca²⁺]_i, myosin light chain (MLC) phosphorylation and force. This resulted in a large increase in the aequorin/Fura-2 ratio, suggesting an increase in [Ca²⁺] inhomogeneity. Addition of 100 μM histamine to tissues in the choline⁺ buffer initially increased both aequorin and Fura-2 estimated [Ca²⁺]_i but after 10 min exposure both of the [Ca²⁺]_i estimates declined to pre-histamine levels. Histamine addition significantly increased MLC phosphorylation and force, indicating increased Ca²⁺ sensitivity, but the aequorin/Fura-2 ratio remained elevated and uncharged from pre-histamine values. These data show that under certain conditions, aequorin and Fura-2 can yield widely differing estimates of [Ca²⁺]_i, and thus can cause misleading assessments of Ca²⁺ sensitization mechanisms. These discrepancies may arise from inhomogeneous or focal increases in [Ca²⁺]_i which can be evaluated with the aequorin/Fura-2 ratio.     

Uptake of liposomes containing the photoprotein obelin by rat isolated adipocytes. Adhesion,endocytosis or fusion?

1. The uptake of liposomes containing the photoprotein obelin by rat isolated adipocytes was investigated with the aim of producing liposome–cell fusion, enabling obelin to be introduced into the cytoplasm of intact cells. 2. Incubation of liposomes containing obelin with rat isolated adipocytes resulted in a time-dependent uptake of entrapped obelin by the adipocytes. The uptake by adipocytes (at 2h) of liposomes prepared from phosphatidylcholine, phosphatidylcholine+phosphatidylserine (molar ratio 4:1) and phosphatidylcholine+N-octadecylamine (molar ratio 4:1) was approx. 6, 10 and 10% of original entrapped obelin per g dry wt. of adipocytes respectively. 3. During incubation with adipocytes some of the liposomes became permeable to Ca²⁺ ions, resulting in stimulation of obelin luminescence from within the liposomes. This increase in permeability to Ca²⁺ seemed to be the result of the interaction of liposomes with the cell membrane. 4. Approx. 50% of liposome uptake could be inhibited by cytochalasin B (500μm). This was consistent with this uptake being the result of endocytosis. The remaining uptake was probably the result of adhesion of liposomes to the cell membrane. 5. In an attempt to detect the presence of cytoplasmic obelin, after incubation of adipocytes with liposomes, a method of causing a rapid rise in cell-membrane permeability to Ca²⁺ was developed in which an anti-cell anti-body–complement reaction occurred at the cell membrane. There was no detectable transfer of active obelin into the cell cytoplasm. 6. After incubation of liposomes with adipocytes in the absence of bovine serum albumin, obelin luminescence from a small proportion of liposomes increased (approx. 1.5%) in response to anti-(5′-nucleotidase) antibody plus complement. 7. It was concluded that under the conditions of these experiments, (a) no detectable transfer (<0.1%) of liposome-entrapped obelin to the adipocyte cytoplasm had occurred, (b) an increase in liposome permeability to Ca²⁺ occurred during incubation with adipocytes, (c) at least 50% of liposome uptake by adipocytes was the result of endocytosis, presumably into secondary lysosomes, and the remaining uptake was apparently the result of loose attachment of liposomes to the cell surface, and (d) in the absence of bovine serum albumin, a portion of at least one surface antigen, the ectoenzyme 5′-nucleotidase, was transferred from the adipocyte membrane to the liposome membrane.     

Intracellular targeting of the photoprotein aequorin: A new approach for measuring,in living cells,Ca<Superscript>2+</Superscript> concentrations in defined cellular compartments

We here present a novel method, based on the targeting of the photoprotein aequorin, for measuring the concentration of Ca²⁺ ions in defined cellular compartments of intact cells. In this contribution we will discuss the application to mitochondria. A chimaeric cDNA was constructed by fusing in frame the aequorin cDNA with that for a mitochondrial protein. The cDNA encoded a “mitochondrially-targeted” aequorin, composed of a typical mitochondrial targeting signal at the N-terminus and the photoprotein at the C-terminus. The cDNA, inserted in the expression vector pMT2, was co-transfected into bovine endothelial and HeLa cells together with the selectable plasmid pSV2-neo and stable transfectants, selected for high aequorin production, were analyzed. In subcellular fractionations, aequorin was shown to be localized in mitochondria; in intact cells, the first direct measurement of mitochondrial free Ca²⁺, [Ca²⁺]_m, were obtained, which showed that [Ca²⁺]_m is low at rest (<0.5 μM), but rapidly increases to the micromolar range upon cell stimulation [1]. These data indicate that mitochondria “sense” very accurately the cytosolic Ca²⁺ concentration ([Ca²⁺]_i), and after cell stimulation [Ca²⁺]_m rises to values capable of activating the Ca²⁺-sensitive mitochondrial dehydrogenases.     

Intracellular calcium release and the mechanisms of parthenogenetic activation of the sea urchin egg.

Parthenogenetic activation of Lytechinus pictus eggs can be monitored after injection with the Ca-sensitive photoprotein aequorin to estimate calcium release during activation. Parthenogenetic treatments, including the nonelectrolyte urea, hypertonic sea water, and ionophore A23187, all acted to release Ca²⁺ from intracellular stores. Ionophore and urea solutions release Ca²⁺ from the same intracellular store as normal fertilization. This intracellular store can be reloaded after 40 min and discharged again. Hypertonic medium appears to release Ca²⁺ from a different intracellular store. Treatment with the weak base NH₄Cl did not release intracellular Ca²⁺ but did result in a momentary Ca²⁺ influx if Ca²⁺ was present in the external solution. Ca²⁺ influx was not required for ammonia activation.     

Overexpression of Sly41 suppresses COPII vesicle–tethering deficiencies by elevating intracellular calcium levels

SLY41 was identified as a multicopy suppressor of loss of Ypt1, a Rab GTPase essential for COPII vesicle tethering at the Golgi complex. SLY41 encodes a polytopic membrane protein with homology to a class of solute transporter proteins, but how overexpression suppresses vesicle-tethering deficiencies is not known. Here we show that Sly41 is efficiently packaged into COPII vesicles and actively cycles between the ER and Golgi compartments. SLY41 displays synthetic negative genetic interactions with PMR1, which encodes the major Golgi-localized Ca²⁺/Mn²⁺ transporter and suggests that Sly41 influences cellular Ca²⁺ and Mn²⁺ homeostasis. Experiments using the calcium probe aequorin to measure intracellular Ca²⁺ concentrations in live cells reveal that Sly41 overexpression significantly increases cytosolic calcium levels. Although specific substrates of the Sly41 transporter were not identified, our findings indicate that localized overexpression of Sly41 to the early secretory pathway elevates cytosolic calcium levels to suppress vesicle-tethering mutants. In vitro SNARE cross-linking assays were used to directly monitor the influence of Ca²⁺ on tethering and fusion of COPII vesicles with Golgi membranes. Strikingly, calcium at suppressive concentrations stimulated SNARE-dependent membrane fusion when vesicle-tethering activity was reduced. These results show that calcium positively regulates the SNARE-dependent fusion stage of ER–Golgi transport.     

The effect of physiologically occurring cations upon aequorin light emission. Determination of the binding constants.

1. The effect of K+, Na+, Mg2+ and pH upon the rate of aequorin utilization has been investigated in the presence of Ca2+. 2. The aequorin light emission in a medium simulating the in vivo cationic conditions for barnacle muscle fibres indicates that two Ca2+ are apparently involved in this process for free calcium concentrations higher than approx. 10(-5) M. However, for free calcium concentrations lower than 10(-6) M, the intensity of light emitted by aequorin shows a steeper dependency upon [Ca2+] than the square low relationship, indicating that a third Ca2+ should be involved in the process of aequorin light emission, as it has been previously predicted (Moisescu, D.G., Ashley, C.C. and Campbell, A.K. (1975) Biochim. Biophys. Acta. 396, 133-140). 3. The inhibitory effect of physiologically occurring cations upon the aequorin light emission can be explained by the cooperative action of two cations, competing with Ca2+ for the reactive sites on aequorin. 4. At a given concentration, Na2+ was found to have a stronger inhibitory effect upon the aequoring light emission than K+. 5. The experiments indicate a strong interaction between Na+ and K+ in this inhibitory process, since for a given total concentration of monovalent cations, a mixture containing both Na+ and K+ has a larger inhibitory effect on the aequorin light response than solutions containing either Na+ or K+ alone. 6. All other interactions between K+, Na+, H+ and Mg2+ appear to be weak. 7. The reaction schemes used for the explanation of these and other published results on aequorin (Moisescu, D.G., Ashley, C.C. and Campbell, A.K. (1975) Biochim. Biophys, Acta 396, 133-140 and Blinks, J.R. (1973) Eur. J. Cardiol. 1, 135-142) are described, and the 'absolute' binding constants of all physiologically occurring cations for aequorin have been determined. 8. Based on these parameters one can make accurate quantitative predictions for the aequoring light response under a variety of ionic conditions, and this suggests that it is possible to determine absolute free calcium concentrations providing that the ionic composition of the solutions is known, and that the relative rate of aequorin utilization is higher than 0.005.     

Simultaneous measurement of aggregation, secretion, oxygen uptake, proton production, and intracellular metabolites in the same platelet suspension.

A cuvette is described for the simultaneous measurement of hydrogen ions, calcium ions, light transmission, and pO₂ in a single gel-filtered platelet suspension stirred at 37°C. A variable volume (2.5–10 ml) and special in- and outlets permit additions and sampling during incubation. Evidence is presented that changes in these parameters reflect lactate production in resting platelets (H⁺ measurement), secretion of dense granular content (Ca²⁺ measurement), shape change and aggregation (light transmission), and arachidonate oxidation and mitochondrial respiration (pO₂ measurement). The cuvette therefore enables direct correlation between function and energy metabolism in platelets.     

Changes in Intracellular Free Calcium Concentration during Illumination of Invertebrate Photoreceptors : Detection with Aequorin

Aequorin, which luminesces in the presence of calcium, was injected into photoreceptor cells of Limulus ventral eye. A bright light stimulus elicited a large increase in aequorin luminescence, the aequorin response, indicating a rise of intracellular calcium ion concentration, Ca_i. The aequorin response reached a maximum after the peak of the electrical response of the photoreceptor, decayed during a prolonged stimulus, and returned to an undetectable level in the dark. Reduction of Ca_o reduced the amplitude of the aequorin response by a factor no greater than 3. Raising Ca_o increased the amplitude of the aequorin response. The aequorin response became smaller when membrane voltage was clamped to successively more positive values. These results indicate that the stimulus-induced rise of Ca_i may be due in part to a light-induced influx of Ca and in part to release of Ca from an intracellular store. Our findings are consistent with the hypothesis that a rise in Ca_i is a step in the sequence of events underlying light-adaptation in Limulus ventral photoreceptors. Aequorin was also injected into photoreceptors of Balanus. The aequorin responses were similar to those recorded from Limulus cells in all but two ways: (a) A large sustained aequorin luminescence was measured during a prolonged stimulus, and (b) removal of extracellular calcium reduced the aequorin response to an undetectable level.