The interaction of calcium and procaine with hepatocyte and hepatoma tissue culture cell plasma membranes studied by fluorescence spectroscopy

The fluorescent probe l-anilinonaphthalene-8-sulfonate (ANS) has been used to investigate the properties of plasma membranes derived from normal hepatocytes and from hepatoma tissue culture (HTC) cells as well as used to study the effects of Ca²⁺ and procaine on these membrane systems. The interaction of ANS with hepatocyte plasma membranes (50 nmol/mg protein; K_D = 120,μM) resulted in a marked enhancement of fluorescence and a 20-nm blue shift. Both Ca²⁺ and procaine further increased the fluorescence intensity. Binding studies showed no alteration in the number of ANS binding sites but a significant decrease in K_D (40–50 μm). Procaine was also shown to completely displace Ca²⁺ from the membrane. The interaction of ANS with HTC cell plasma membranes again resulted in an enhancement in fluorescence intensity but with different binding properties (102 nmol/mg protein; K_D = 74 μM) from the hepatocyte system. The addition of Ca⁺² resulted in the formation of high and low affinity ANS binding sites as shown by Scatchard plot analysis with K_D values of 15 μm and 50 μm. The effect of procaine on ANS fluorescence in the normal and transformed cell membranes was indistinguishable; however, in the latter system procaine only displaced 60% of the bound Ca²⁺. These studies suggest several structural and binding alterations between plasma membranes derived from hepatocytes and HTC cells.     

Energy transfer from tryptophan residues of proteins to 8-anilinonaphthalene-1-sulfonate

The binding of the apolar fluorescent dye 8-anilinonaphthalene-1-sulfonate (ANS) to bovine serum albumin (BSA), phospholipase A₂ (PLA₂), ovalbumin, lysozyme, cobrotoxin and N-acetyltryptophanamide was used to assess the factors affecting the efficiency of energy transfer from Trp residues to the ANS molecule. We found that the efficiency of energy transfer from Trp residues to ANS was associated with the ability of proteins to enhance the ANS fluorescence. At the same molar concentration of protein, BSA enhanced ANS fluorescence most among these proteins; its Trp fluorescence was drastically quenched by the addition of ANS. Fluorescence enhancement of ANS in PLA₂-ANS complex increased upon addition of Ca²⁺ or change of the buffer to acidicpH, resulting in a higher efficiency of energy transfer from Trp residues to ANS. There was limited ANS fluorescence enhancement with ovalbumin, lysozyme, cobrotoxin, and N-acetyltryptophanamide and a less efficient quenching in Trp fluorescence. The capabilities of proteins for binding with ANS correlated with the decrease in their Trp fluorescence being quenching by ANS. However, the microenvironment surrounding Trp residues of proteins did not affect the energy transfer. Based on these results, the factors that affected the energy transfer from Trp residues to ANS are discussed.     

Enhancement of salt responses in frog gustatory nerve by removal of Ca2+ from the receptor membrane treated with 1-anilinonaphthalene-8-sulfonate

Summary The frog tongue was incubated in 1-anilinonaphthalene-8-sulfonate (ANS) solution and the responses of the glossopharyngeal nerve to various chemical stimuli were measured after the ANS solution was washed out. The responses to galactose, quinine and distilled water were unchanged by the ANS treatment. On the other hand, the responses to the salts, except for CaCl₂, were enhanced in greater or lesser degree after the ANS treatment. The order of relative magnitude of the enhanced response to 100mm salts of monovalent cations was Na⁺>NH ₄ ⁺ >K⁺>Li⁺, while that before the treatment was NH ₄ ⁺ >K⁺>Na⁺>Li⁺. The enhancement of the salt responses was also observed after the tongue was treated with 6-p-toluidinonaphthalene-2-sulfonate or 1,2-cyclohexanediaminetetraacetic acid solution.The enhanced responses to the salts were suppressed to the original level before the ANS treatment by addition of CaCl₂ or SrCl₂. The suppression curve satisfied the Langmuir adsorption isotherm when the suppression was postulated to be responsible for the binding of Ca²⁺ or Sr²⁺ to the receptor membrane treated with ANS. The apparent binding constants for Ca²⁺ and Sr²⁺ in the presence of 100mm NaCl were obtained to be 1.2×10⁴ m ^–1 and 6.7×10³ m ^–1, respectively.The ANS treatment modified the temperature dependence of the salt responses. For example, 100mm KCl solution of low temperature induced a large response after the ANS treatment, while that of 20°C induced only small response.It was concluded that the removal of Ca²⁺ from the gustatory receptor membrane in the frog, which was brought about by the ANS treatment, led to the enhancement of the salt responses. The mechanism on the enhancement of the salt response by the Ca²⁺ removal was discussed.     

The essentiality of calcium ion in the enzymatic activity of Taiwan cobra phospholipase A2

In order to address the mechanism whereby Ca²⁺ wad crucial for the manifestation of the enzymatic activity of phospholipase A₂ (PLA₂), four divalent cations were used to assess their influences on the catalytic activity and the fine structures ofNaja naja atra PLA₂. It was found that substitution of Mg²⁺ or Sr²⁺ for Ca²⁺ in the substrate solution caused a decrease in the PLA₂ activity to 77.5% or 54.5%, respectively, of that in the presence of Ca²⁺. However, no PLA₂ activity was observed with the addition of Ba²⁺. With the exception of Mg²⁺, the nonpolarity of the 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of PLA₂ markedly increased with the binding of cations to PLA₂. In the meantime, the accessibilities of Lys-6 (65) and Tyr-3 (63) toward trinitrobenzene sulfonate andp-nitrobenzenesulfonyl fluoride were enhanced by the addition of Ca²⁺, Sr²⁺, and Ba²⁺, but not by Mg²⁺. The order of the ability of cations to enhance the ANS fluorescence and the reactivity of Lys and Tyr residues toward modified reagents was Ba²⁺> Sr²⁺> Ca²⁺> Mg²⁺, which was the same order as the increase in their atomic radii. These results, together with the observations that the ANS molecule binds at the active site of PLA₂ and that Tyr-3, Lys-6, and Tyr-63 of PLA₂ are involved in the binding with the substrate, suggest that the binding of Ca²⁺ to PLA₂ induces conformational changes at the active site and substrate-binding site. However, the smaller atomic radius with Mg²⁺ or the bigger atomic radii with Sr²⁺ and Ba²⁺ might render the conformation improperly rearranged after their binding to PLA₂ molecule.     

Mechanism of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> death induced by <Emphasis Type="Italic">Cratylia mollis</Emphasis> seed lectin

Incubation of T. cruzi epimastigotes with the lectin Cramoll 1,4 in Ca²⁺ containing medium led to agglutination and inhibition of cell proliferation. The lectin (50 μg/ml) induced plasma membrane permeabilization followed by Ca²⁺ influx and mitochondrial Ca²⁺ accumulation, a result that resembles the classical effect of digitonin. Cramoll 1,4 stimulated (five-fold) mitochondrial reactive oxygen species (ROS) production, significantly decreased the electrical mitochondrial membrane potential (ΔΨ_m) and impaired ADP phosphorylation. The rate of uncoupled respiration in epimastigotes was not affected by Cramoll 1,4 plus Ca²⁺ treatment, but oligomycin-induced resting respiration was 65% higher in treated cells than in controls. Experiments using T. cruzi mitochondrial fractions showed that, in contrast to digitonin, the lectin significantly decreased ΔΨ_m by a mechanism sensitive to EGTA. In agreement with the results showing plasma membrane permeabilization and impairment of oxidative phosphorylation by the lectin, fluorescence microscopy experiments using propidium iodide revealed that Cramoll 1,4 induced epimastigotes death by necrosis.     

The uptake of calcium by isolated chromaffin granules of the adrenal medulla

Bovine chromaffin secretory granules were purified by isopycnic Metrizamide gradient centrifugation and their Ca²⁺ sequestration pathways were characterized. The rate of Ca²⁺ sequestration at 37°C was first order, with a maximal uptake of 26.9 ±0.46 (mean ± S.D., n = 3) nmol Ca²⁺/mg protein and a first order rate constant (k) of 0.046 ± 0.002 min^–1. At 4°C the rate of uptake was substantially attenuated, with only 2.47 ± 0.2 (mean ± S.D, n = 3) nmol Ca²⁺/mg protein sequestered in 60 min. Ca²⁺ sequestration was 93% inhibited by 180 mM NaCl [I_50% of 78.7 ± 9.3 mM NaCl (mean ± S.D., n = 11)] but only slightly inhibited by KCl or MgCl₂. Ca ²⁺ sequestration was not stimulated by incubation with MgATP but was inhibited by 57% after incubation with 30 M monensin. Ca ²⁺ sequestration was dependent on extravesicular Ca ²⁺ with half-maximal sequestration at pCa²⁺ 6.81 ± 0.028 (mean ± S.D., n = 3). Sequestered Ca²⁺ could be exchanged with external ⁴⁵Ca²⁺, the exchange rate was first order (k of 0.042 ± 0.004: mean ± S.D., n = 3) and saturated at 27.7 ± 1.1 nmol Ca²⁺/mg (mean ± S.D., n = 3). The Ca²⁺/Ca²⁺ exchange system was totally inhibited by NaCl or KCl but only slightly by MgCl₂. About 75% of sequestered ⁴⁵Ca²⁺ could be released by incubation with NaCl, but only 8% was released by incubation with KCI. Half-maximal release of sequestered ⁴⁵Ca²⁺ required 69.3 ± 12.2 mM NaCl (mean ± S.D., n = 3). The Na⁺-induced release of sequestered ⁴⁵Ca²⁺ was rapid, t_0.5 of 2.80 ± 0.63 min (mean ± S.D., n = 3) and inhibited at 4°C. The concurrent incubation of chromaffin granules with ⁴⁵Ca²⁺ and either annexin proteins V or VI resulted in attenuated uptake of ⁴⁵Ca²⁺. These results suggest that Ca²⁺ uptake in adrenal chromaffin granules is regulated by Na⁺ and Ca²⁺ gradients and also possibly by annexins V and VI.Abbreviations EGTA ethylene glycol bis (-aminoethyl ether)-N,-N,N,N-tetraacetic acid - SDS Sodium dodecyl sulphate - PAGE Polyacrylamide gel electrophoresis - BSA bovine serum albumin - AI Annexin I - AIIt Annexin II tetramer - AIII Annexin III - AIV Annexin IV - AV Annexin V - AVI Annexin VI - k first order rate constant - AT total extent of Ca²⁺ uptake (nmol) - BufferA 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 5 mM EGTA - Buffer B 300 mM sucrose, 10 mM potassium phosphate (pH 7.0) and 1 mM EGTA - Buffer C 300 mM sucrose, 10 mM potassium phosphate (pH 7.0) - Buffer D 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.5 mM EGTA and 0.65 MM CaCl₂ - Buffer E 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.25 mM EGTA and 0.325 mM CaCl₂     

Efflux of magnesium and potassium ions from liver mitochondria induced by inorganic phosphate and by diamide

Addition to rat liver mitochondria of 2 mM inorganic phosphate or 0.15 mM diamide, a thiol-oxidizing agent, induced an efflux of endogenous Mg²⁺ linear with time and dependent on coupled respiration. No net Ca²⁺ release occurred under these conditions, while a concomitant release of K⁺ was observed. Mg²⁺ efflux mediated either by P_i or low concentrations of diamide was completely prevented by EGTA, Ruthenium red, and NEM. These reagents also inhibited the increased rate of state 4 respiration induced both by P_i and diamide. At higher concentrations (0.4 mM), diamide induced an efflux of Mg²⁺ which was associated also with a release of endogenous Ca²⁺. Under these conditions EGTA completely prevented Mg²⁺ and K⁺ effluxes, while they were only partially inhibited by Ruthenium red and NEM. It is assumed that Mg²⁺ efflux, occurring at low diamide concentrations or in the presence of phosphate, is dependent on a cyclic in-and-out movement of Ca²⁺ across the inner mitochondrial membrane, in which the passive efflux is compensated by a continuous energy linked reuptake. This explains the dependence of Mg²⁺ efflux on coupled respiration, as well as the increased rate of state 4 respiration. The dependence of Mg²⁺ efflux on phosphate transport is explained by the phosphate requirement for Ca²⁺ movement.Abbreviations Diamide diazenedicarboxylic acidbis-dimethylamide - FCCP p-trifluoromethoxyphenylhydrazone - EGTA ethylene glycol-bis-(2-amino ethyl ether)-N,N-tetracetic acid - P_i inorganic phosphate - Ruthenium red Ru₂(OH)₂Cl₄ · 7NH₃ · 3H₂O - state 4 controlled state of respiration in the presence of substrate - RCI respiratory control index - NEM N-ethyl maleimide A partial and preliminary report of these results has been published inBiochem. Biophys. Res. Comm.,78 (1977) 23.     

Ruthenium red-sensitive and Ruthenium Red-insensitive release of calcium by mitochondria isolated from rat liver and from rat heart

EGTA (ethanedioxybis(ethylamine)tetra-acetic acid) induced a release of Ca²⁺ from mitochondria isolated from both rat liver and rat heart that was inhibited by Ruthenium Red. The concentration of Ruthenium Red giving half-maximal inhibition was about 350 pmol/mg of protein, a value approximately 7 times greater than that giving half-maximal inhibition of the initial rate of Ca²⁺ transport. The EGTA-induced release of Ca²⁺ was temperature-dependent and was inhibited by the local anaesthetic, nupercaine.Pi, acetate, and tributyltin in the presence of Cl^?, inhibited the Ruthenium Red-sensitive Ca²⁺ release induced by EGTA, whereas these agents enhanced the Ruthenium Red-insensitive release of Ca²⁺ induced by acetoacetate in liver and heart mitochondria and by Na⁺ in heart mitochondria.     

Ca binding to sarcoplasmic reticulum ATPase phosphorylated by Pi reveals four thapsigargin-sensitive Ca sites in the presence of ADP

Sarcoplasmic reticulum (SR) Ca²⁺-ATPase was phosphorylated by P_i at pH 8.0 in the presence of dimethyl sulfoxide (Me₂SO). Under these conditions, it was possible to measure transient ⁴⁵Ca²⁺ binding to the phosphoenzyme. Binding reached 1.2 Ca²⁺ per phosphoenzyme (E-PCa_x) within 10 min in 30% Me₂SO, 20 mM MgCl₂ and 0.1 mM P_i and the phosphoenzyme only decreased by 23% during this period. This Ca²⁺ binding was abolished by thapsigargin, showing that it is associated with functional sites of the Ca²⁺-ATPase. At 40% Me₂SO, simultaneous addition of Ca²⁺ and ADP increased Ca²⁺ binding up to almost four Ca²⁺ per phosphoenzyme (ADPE-PCa_y), revealing a species bearing simultaneously four Ca²⁺ sites. Both E-PCa_x and ADPE-PCa_y were further identified as distinct species by (2′,3′-O-2(2,4,6-trinitrophenyl)adenosine 5′-triphosphate) fluorescence, which revealed long-range modifications in the Ca²⁺-transport sites induced by ADP binding to E-P. In addition, E-PCa_x was shown to be a functional intermediate of the cycle leading to ATP synthesis provided that Me₂SO was diluted. These findings indicate that more than two functional Ca²⁺-sites exist on the functional Ca²⁺-ATPase unit, and that the additional sites become accessible upon ADP addition. This is compatible with a four-site model of the SR Ca²⁺-ATPase allowing simultaneous binding of Ca²⁺ at lumenal and cytosolic sites. The stoichiometries for Ca²⁺ binding found here could either be interpreted as binding of four Ca²⁺ on a Ca²⁺-ATPase monomer considered as the functional unit or as binding of two Ca²⁺ per monomer of a functional dimer.     

P2Y13 Receptor-Mediated Rapid Increase in Intracellular Calcium Induced by ADP in Cultured Dorsal Spinal Cord Microglia

P2Y receptors have been implicated in the calcium mobilization by the response to neuroexcitatory substances in neurons and astrocytes, but little is known about P2Y receptors in microglia cells. In the present study, the effects of ADP on the intracellular calcium concentration ([Ca²⁺]i) in cultured dorsal spinal cord microglia were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescence indicator that could monitor real-time alterations of [Ca²⁺]i. Here we show that ADP (0.01–100 μM) causes a rapid increase in [Ca²⁺]i with a dose-dependent manner in cultured microglia. The action of ADP on [Ca²⁺]i was significantly blocked by MRS2211 (a selective P2Y₁₃ receptor antagonist), but was unaffected by MRS2179 (a selective P2Y₁ receptor antagonist) or MRS2395 (a selective P2Y₁₂ receptor antagonist), which suggest that P2Y₁₃ receptor may be responsible for ADP-evoked Ca²⁺ mobilization in cultured microglia. P2Y₁₃-evoked Ca²⁺ response can be obviously inhibited by BAPTA-AM and U-73122, respectively. Moreover, removal of extracellular Ca²⁺ (by EGTA) also can obvious suppress the Ca²⁺ mobilization. These results means both intracellular calcium and extracellular calcium are potentially important mechanisms in P2Y₁₃ receptor-evoked Ca²⁺ mobilization. However, P2Y₁₃ receptor-evoked Ca²⁺ response was not impaired after CdCl₂ and verapamil administration, which suggest that voltage-operated Ca²⁺ channels may be not related with P2Y₁₃-evoked Ca²⁺ response. In addition, Ca²⁺ mobilization induced by ADP was abolished by different store-operated Ca²⁺ channels (SOCs) blocker, 2-APB (50 μM) and SKF-96365 (1 mM), respectively. These observations suggest that the activation of P2Y₁₃ receptor might be involved in the effect of ADP on [Ca²⁺]i in cultured dorsal spinal cord microglia. Furthermore, our results raise a possibility that P2Y₁₃ receptor activation causes Ca²⁺ release from Ca²⁺ store, which leads to the opening of SOCs.     

The thermodynamic efficiency of the Ca2+-Mg2+-ATPase is one hundred percent

The thermodynamic efficiency of the Ca²⁺-Mg²⁺-ATPase of skeletal sarcoplasmic reticulum has been evaluated by comparing the Ca²⁺ gradient established with the ATP/(ADP*P_i) ratio. The evaluation was made at an external Ca²⁺ level (4.7 × 10^–8 M) which is below theK _m value of 7 × 10^–8 M. The Mg-ATP and phosphate concentrations were held constant (0.1 mM) and the ADP concentration was varied. Maximal uptake to an internal free Ca²⁺ concentration of 17 mM was observed at infinite ATP/(ADP*P_i) ratio (absence of ADP). This corresponds to a [Ca²⁺]_i/[Ca²⁺]₀ gradient of 3.6 × 10⁵. A Ca²⁺ gradient one-half as large was observed at an ATP/(ADP*P_i) ratio of 3.5 × 10³ M^–1. The square of the Ca²⁺ gradient is shown to be proportional to the ATP/(ADP*P_i) ratio, for finite values of the latter. The proportionality constant is identical to the equilibrium constant for hydrolysis of ATP (9.02 × 10⁶ M) under these conditions (0.1 mM Mg²⁺, 30°C). The intrinsic thermodynamic efficiency of the pump is shown to be 100%, with a maximal uncertainty of 3%. The efficiency is lower under less optimal conditions, when the pump is inhibited and passive leak processes compete.Dedicated to Prof. Philip George, University of Pennsylvania, whose instruction, research, and example made this contribution possible.     

The essentiality of calcium ion in the enzymatic activity of Taiwan cobra phospholipase A2

In order to address the mechanism whereby Ca²⁺ wad crucial for the manifestation of the enzymatic activity of phospholipase A₂ (PLA₂), four divalent cations were used to assess their influences on the catalytic activity and the fine structures ofNaja naja atra PLA₂. It was found that substitution of Mg²⁺ or Sr²⁺ for Ca²⁺ in the substrate solution caused a decrease in the PLA₂ activity to 77.5% or 54.5%, respectively, of that in the presence of Ca²⁺. However, no PLA₂ activity was observed with the addition of Ba²⁺. With the exception of Mg²⁺, the nonpolarity of the 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of PLA₂ markedly increased with the binding of cations to PLA₂. In the meantime, the accessibilities of Lys-6 (65) and Tyr-3 (63) toward trinitrobenzene sulfonate andp-nitrobenzenesulfonyl fluoride were enhanced by the addition of Ca²⁺, Sr²⁺, and Ba²⁺, but not by Mg²⁺. The order of the ability of cations to enhance the ANS fluorescence and the reactivity of Lys and Tyr residues toward modified reagents was Ba²⁺> Sr²⁺> Ca²⁺> Mg²⁺, which was the same order as the increase in their atomic radii. These results, together with the observations that the ANS molecule binds at the active site of PLA₂ and that Tyr-3, Lys-6, and Tyr-63 of PLA₂ are involved in the binding with the substrate, suggest that the binding of Ca²⁺ to PLA₂ induces conformational changes at the active site and substrate-binding site. However, the smaller atomic radius with Mg²⁺ or the bigger atomic radii with Sr²⁺ and Ba²⁺ might render the conformation improperly rearranged after their binding to PLA₂ molecule.     

Tryptophan modification of phospholipase A2 enzymes and presynaptic neurotoxins from snake venoms

Three phospholipases A₂ purified from cobra venoms and two presynaptically acting neurotoxins that exhibit phospholipase A₂ activity were subjected to tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Associated with the modification of an increasing number of Trp residues were marked decreases in enzymatic activity and lethality, whereas antigenicity remained unchanged. The degree of exposure of tryptophanyl groups as determined by acrylamide quenching was consistent with the relative reactivity toward 2-hydroxy-5-nitrobenzyl bromide, except for Hemachatushaemachatus phospholipase A₂, which showed unusually high reactivity due to its characteristic dimeric conformation. Difference spectra of Trp-modified derivatives differed from those of their native enzymes by the presence of a new positive perturbation between 350 and 500 nm, with a maximum at 415 nm. Scatchard plots revealed only one type of binding site for Ca²⁺, and the binding abilities of the modified enzymes were not impaired. At pH 8.0, all native enzymes enhanced the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, and the emission intensity of the ANS-enzyme complex increased or decreased in parallel with increasing concentration of Ca²⁺ for the respective enzyme. The Trp-modified derivatives did not enhance the emission intensity of ANS at all either in the presence or absence of Ca²⁺. By means of tryptophan modification, we were able to infer that the tryptophan residues are in the vicinity of the Ca²⁺ binding site and are directly involved in the binding with ANS. This, together with the suggestion that the hydrophobic pocket that interacts with ANS might be the site of binding of the phospholipase A₂ enzyme with the substrate, suggests that the Trp residues in phospholipase A₂ enzymes and presynaptic toxins are involved in substrate binding.     

Rapid kinetic study of the passive permeability of a Ca2+-ATPase rich fraction of the sarcoplasmic reticulum

Summary A method was developed for the study of divalent cation transport events on the time scale of 20 msec or longer. Passive Ca²⁺ equilibration across the membranes of the Ca²⁺-ATPase rich fraction of sarcoplasmic reticulum (SR) was studied. The method makes use of the divalent cation sensitivity of the surface binding of the fluorescent probe 1-anilino-8-naphthalenesulfonate (ANS^–). Binding to the inside and outside surfaces is distinguished in fluorescent stopped-flow experiments. The surface binding reactions of the probe are faster than the time resolution of the instrument (ca. 3 msec), while binding reactions requiring transport across the membrane could be resolved. In Ca²⁺ influx experiments, the time course of fluorescent enhancement was monitored following a Ca²⁺ jump. The kinetics of Ca²⁺ efflux were studied by pre-equilibrating Ca²⁺ across the membrane, removing the external Ca²⁺ with an EGTA jump, and observing the time course of the fluorescence decrease. Rapid transport of ANS^– (coupled to K⁺) was ensured by the addition of valinomycin. Two processes of Ca²⁺ influx were observed: (i) a rapid process with small fluorescent amplitudes and at _1/2 of 40–60 msec and (ii) a slow process with a large amplitude and at _1/2 of 70–100 sec. The rates and extents of the two phases were quantitated in terms of the rates and extents of change in the Ca²⁺ concentration in the SR lumen. The slow phase accounted for a larger change, in the internal free Ca²⁺ concentration than did the first phase. For the influx of 10 mM Ca²⁺, the rapid phase raises the internal Ca²⁺ concentration to ca. 1 mM within its apparentt _1/2 of 20 msec. The slow phase brings about an increase of the internal Ca²⁺ concentration to 4 mM within its apparentt _1/2 of 90 sec. The two phases have average rates of increase of internal free Ca²⁺ concentration, [Ca] _i /sec of ca. 50 mM/sec and ca. 0.02 mM/sec, respectively. The Ca²⁺ influx rates increased with increasing KCl concentration and with increasing external Ca²⁺ concentration.Two phases of Ca²⁺ efflux were observed. The amplitudes and rates were analyzed and the fast phase was shown to account for more Ca²⁺ movement than the slow phase. The rate of the fast phase was greatly increased by increasing the K⁺ concentration. The rate of the slow phase efflux decreased with increasing Ca²⁺ concentration in the external medium. The concentration for half-maximal inhibition was 4 M, a value close to the dissociation constant of the high affinity site on the Ca²⁺-ATPase.The above constitutes a body of circumstantial evidence that the passive Ca²⁺ permeability observed is mediated by the Ca²⁺-ATPase, acting as a Ca²⁺ for 2 K⁺ exchanger. The fast phases are explained as a partial turnover of the pump in the steady state. The slow rate is explained by a preference of the ion binding translocator site of the carrier for an outward orientation.The ANS^– technique was applied to the monovalent cation permeability of the Ca²⁺-ATPase rich SR and the results of other studies were corroborated and extended. The interaction of valinomycin with intrinsic permeability mechanisms of the SR was considered.     

Direct inhibitory action of EGTA-Ca complex on reverse-mode Na/Ca exchange inMyxicola giant axons

Summary Giant axons from the marine annelidMyxicola infundibulum were internally dialyzed with solutions containing²²Na ions as tracers of Na efflux. In experiments performed in Li-substituted seawater, Na efflux that is dependent on external Ca ion concentration, [Ca²⁺] _o , was measured using dialysis to maintain [Na⁺] _i at 100mm, which enhances the [Ca²⁺] _o -dependent Na efflux component, (i.e., reverse-mode Na/Ca exchange). When dialysis fluid contained EGTA (1mm) to buffer the internal Ca concentration, [Ca²⁺] _i , to desired levels, Na efflux lost its normal sensitivity to external calcium. The inhibition was not simply due to the Ca-chelating action of EGTA to produce insufficient [Ca²⁺] _i to activate Na/Ca exchange. The addition of EGTA inhibited Ca _o -dependent Na efflux even when a large enough excess of [Ca²⁺] _i was present to saturate the EGTA and still produce elevated values of [Ca²⁺] _i . Control experiments showed that these high values of [Ca²⁺] _i resulted in normal Na/Ca exchange in the absence of EGTA. It is concluded that the presence of EGTA itself interferes with the manifestation of reverse-mode Na/Ca exchange inMyxicola giant axons.     

Calcium is involved in the abscisic acid-induced ascorbate peroxidase, superoxide dismutase and chilling resistance in Stylosanthes guianensis

The objective of this work was to test whether Ca²⁺, a second messenger in stress response, is involved in ABA-induced antioxidant enzyme activities in Stylosanthes guianensis. Plants were sprayed with abscisic acid (ABA), calcium channel blocker, LaCl₃, calcium chelator, ethylene glycol-bis(β-amino ethyl ether)-N,N,N′, N′-tetraacetid acid (EGTA), and ABA in combination with LaCl₃ or EGTA. Their effects on superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities and chilling resistance were compared. The results showed that ABA decreased electrolyte leakage and lipid peroxidation but increased maximum photochemical efficiency measured as variable to maximum fluorescence ratio (F_v/F_m) under chilling stress. Treatment with LaCl₃ or EGTA alone and in combination with ABA increased electrolyte leakage and lipid peroxidation, decreased Fv/Fm, suggesting that the block in Ca²⁺ signalling decreased chilling resistance of S. guianensis and the ABA-enhanced chilling resistance. ABA-induced SOD and APX activities were suppressed by LaCl₃ or EGTA. The results suggested that Ca²⁺ is involved in the ABA-enhanced chilling resistance and the ABA-induced SOD and APX activities in S. guianensis.     

K+-and Mg2+-dependent hydrolysis of acetyl phosphate catalyzed by the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

The (Ca²⁺ + Mg²⁺-ATPase of sarcoplasmic reticulum catalyzes the hydrolysis of acetyl phosphate in the presence of Mg²⁺ and EGTA and is stimulated by Ca²⁺. The Mg²⁺-dependent hydrolysis of acetyl phosphate measured in the presence of 6 mM acetyl phosphate, 5mM MgCl₂, and 2 mM EGTA is increased 2-fold by 20% dimethyl sulfoxide. This activity is further stimulated 1.6-fold by the addition of 30 mM KCl. In this condition addition of Ca²⁺ causes no further increase in the rate of hydrolysis and Ca²⁺ uptake is reduced to a low level. In leaky vesicles, hydrolysis continues to be back-inhibited by Ca²⁺ in the millimolar range. Unlike ATP, acetyl phosphate does not inhibit phosphorylation by P_i unless dimethyl sulfoxide is present. The presence of dimethyl sulfoxide also makes it possible to detect P_i inhibition of the Mg²⁺-dependent acetyl phosphate hydrolysis. These results suggest that dimethyl sulfoxide stabilizes a P_i-reactive form of the enzyme in a conformation that exhibits comparable affinities for acetyl phosphate and P_i. In this conformation the enzyme is transformed from a Ca²⁺- and Mg²⁺-dependent ATPase into a (K⁺ + Mg²⁺)-ATPase.     

Intrinsic fluorescence studies on troponin C.

Evidence for a proton transfer mechanism in the Ca²⁺-induced enhancement of the Tyr fluorescence of troponin C was obtained by studying the effects, in D₂O and H₂O, of Ca²⁺, Mg²⁺, and H⁺ on the fluorescence of both the protein and a model system containing L-Tyr in the presence of citrate. In the model system, it is shown that citrate quenches the fluorescence of L-Tyr, that there is a large deuterium isotope effect on the quenching, and that binding of Ca², Mg²⁺, or H⁺ by citrate results in a fluorescence enhancement. These results can be explained by the transfer of the phenolic proton of the excited Tyr to the carboxylates of citrate. Similar effects on the fluorescence of troponin C suggest that, in the apoprotein, the fluorescence of Tyr is quenched by a similar mechanism. Thus, the Ca²⁺, Mg²⁺, and H⁺-induced Tyr fluorescence enhancement in troponin C is due to “dequenching” resulting from coordination or protonation of vicinal carboxylates. Studies of troponin C fluorescence and fluorescence depolarization as a function of urea concentration enabled an estimate of the separate fluorescence contributions of its two Tyr residues (Nos. 10 and 109) in its various conformational states. Further evidence was also obtained to support the earlier proposal that the Ca²⁺ enhancement is primarily due to the direct loss of quenching by nearby carboxylates, by showing that the pH-induced fluorescence enhancement did not occur in parallel with the ellipticity increase at 222 nm.     

Role of calcium and calmodulin antagonist in photosynthesis and salinity tolerance inChlorella vulgaris

To cast light upon the role of Ca¹⁺ and calmodulin on photosynthetic rate (P_n), dark respiration (R_D) and amino acid and protein contents in salinity stressed and non-stressedChlorella cultures, the Ca²⁺ chelator EGTA [ethylene glycol-bis-(2-aminoethyl ether)-N,N- tetraacetate] and the calmodulin antagonist TFP (trifluperazine) were used. TFP markedly inhibited P_N while EGTA exerted a slight, if any, effect on P_N. NaCl tolerance, on the other side, was markedly abolished by TFP that inhibited P_N and lowered rate of proline accumulation. Calmodulin might be involved in osmoregulation and salt tolerance ofChlorella. R_D, however, was markedly enhanced by EGTA and Ca²⁺-free medium and hence the Ca²⁺ deprivation increased stress severity exerted by NaCl. Combinations of Na⁺ and Ca²⁺ enhanced P_N, decreased R_D and proline content in comparison with an osmotically equivalent reference culture containing only NaCl. Addition of Ca²⁺ to TFP treated cultures failed to reactivate calmodulin for proline synthesis. However, when Ca²⁺ was added to EGTA-treated cultures, only relatively reduced proline contents were recorded.     

Study of the mitochondrial phosphate carrier in the course of calcium phosphate accumulation: A requirement for Mg2+ and ADP of its sensitivity to thiol reagents

1. The accumulation of calcium phosphate driven by succinate oxidation is ADP-dependent. In its absence the accumulation stops after a short incubation time and the oxygen uptake is permanently stimulated. This uncoupled oxygen uptake is insensitive to the inhibitors of phosphate transport, like mersalyl and N-ethylmaleimide. When ADP plus Mg²⁺ are added to the medium, or when ADP is added in the initial presence of magnesium, the inhibitory action of the thiol reagents on oxygen uptake is re-established. ADP alone or Mg²⁺ alone are without any effect.2. Phosphate/phosphate exchange has been studied, in the absence of ADP, when calcium phosphate accumulation had stopped and oxygen uptake is uncoupled. Under these conditions the exchange process becomes insensitive to thiol reagents. Sensitivity is recovered solely in the presence of ADP plus Mg²⁺.3. When mitochondrial swelling is studied according to the method of Chappell, it also appears that the phosphate carrier loses it sensitivity to mersalyl in the absence of ADP, which confirms the data obtained with phosphate/phosphate exchange experiments. When ADP plus Mg²⁺ are added (or present), together with mersalyl, the action of the thiol inhibitor is recovered. ADP and magnesium are inactive separately. EGTA plus Mg²⁺ (but not EGTA plus ADP) may substitute for ADP plus Mg²⁺ in this process.4. A possible interaction between the magnesium binding site and the phosphate carrier is considered and discussed.