Ca2+ and Mg2+-binding and a putative calmodulin type Ca2+-binding site in Synechococcus Photosystem II

Thylakoids and Photosystem II particles prepared from the cyanobacterium Synechococcus PCC 7942 washed with a HEPES/glycerol buffer exhibited low rates of light-induced oxygen evolution. Addition of either Ca²⁺ or Mg²⁺ to both thylakoids and Photosystem II particles increased oxygen evolution independently, maximal rates being obtained by addition of both ions. If either preparation was washed with NaCl, light induced O₂ evolution was completely inhibited, but re-activated in the same manner by Ca²⁺ and Mg²⁺ but to a lower level. In the presence of Mg²⁺, the reactivation of O₂ evolution by Ca²⁺ allowed sigmoid kinetics, implying co-operative binding. The results are interpreted as indicating that not only Ca²⁺, but also Mg²⁺, is essential for light-induced oxygen evolution in thylakoids and Photosystem II particles from Synechococcus PC 7942. The significance of the reactivation kinetics is discussed. Reactivation by Ca²⁺ was inhibited by antibodies to mammalian calmodulin, indicating that the binding site in Photosystem II may be analogous to that of this protein.Abbreviation HEPES n-2-Hydroxyethylpiperazine--2-ethane sulphonic acid     

Identification of glycogen as the major xylose acceptor in polysomal preparations from chick embryo cartilage cultures

The capacity of various metal ions to support activation of bovine factor IX, by the coagulant protein of Russell's Viper venom, has been examined. The following metal ions, at concentrations which saturate their effect, promoted activation of factor IX, at approximately equal efficiency: Ca²⁺, Mn²⁺, Sr²⁺, and Co²⁺, Other metal ions, i.e., Ba²⁺, and Mg²⁺, at saturating concentrations, led to a maximum rate of activation of factor IX of 25%, compared to Ca²⁺, The lanthanides, Gd²⁺, and Tb³⁺, also promoted activation in this system, at maximal rates of approximately 15%, compared to Ca²⁺, In this study, it was also discovered that the esterase activity of bovine factor IXa was dependent upon the presence of metal ions. Utilizing α-N-benzoyl-l-arginine ethyl ester as the substrate, steady state kinetic analysis in the absence of metal ion indicated that the K_m and V_max for this substrate was 20 mm and 2.9 μmol substrate cleaved min^?1 mg^?1 of factor IXa, respectively, at pH 8.0 and 30 °C. In the presence of optimal concentrations of Ca²⁺, Mn²⁺, Mg²⁺, Sr²⁺, and Ba²⁺, the V_max values for this same substrate increased to 6.7, 5.9, 5.0, 5.0, and 3.7 μmol cleaved min^?1 mg^?1 of factor IXa, respectively. None of these metal ions had an affect on the K_m value of this substrate.     

Aragonite crystallization in a Christmas-tree model microfluidic device with the addition of magnesium ions

Aragonite is an important dimorph of calcium carbonate, industrially and biologically. However, aragonite is so thermodynamically unstable that it is difficult to understand its formation mechanism. A continuous microfluidic system was employed, in which crystallization was induced only by diffusion in a micron-scale channel. Calcium carbonate (CaCO₃) formed by liquid-liquid reaction and magnesium ions (Mg²⁺) were used as additives. To assess the influence of Mg²⁺ concentration, the Mg²⁺/Ca²⁺ molar ratio was set to 1, 3, and 5. Laminar streams flowed in the detection channel with different concentration gradients. The initial crystallization time (t_I.C) increased exponentially and the density of crystals decreased as the Mg²⁺ ion concentration increased. Following transformation of all particles into snowman or sphere shapes, they became spinose sphere-shaped crystals, which was the final form in this study.     

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.     

Thermostable alkaline phytase from Bacillus sp. MD2: effect of divalent metals on activity and stability

Phytate, the major source of phosphorus in seeds, exists as a complex with different metal ions. Alkaline phytases are known to dephosphorylate phytate complexed with calcium ions in contrast to acid phytases that act only on phytic acid. A recombinant alkaline phytase from Bacillus sp. MD2 has been purified and characterized with respect to the effect of divalent metal ions on the enzyme activity and stability. The presence of Ca²⁺ on both the enzyme and the substrate is required for optimal activity and stability. Replacing Ca²⁺ with Ba²⁺, Mn²⁺, Mg²⁺ and Sr²⁺ in the phytase resulted in the expression of > 90% of the maximal activity with calcium-phytate as the substrate, while Fe²⁺ and Zn²⁺ rendered the enzyme inactive. On the other hand, the calcium loaded phytase showed significant activity (60%) with sodium phytate and lower activity (17-20%) with phytate complexed with only Mg²⁺, Sn²⁺ and Sr²⁺, respectively. On replacing Ca²⁺ on both the enzyme and the substrate with other metal ions, about 20% of the maximal phytase activity was obtained only with Mg²⁺ and Sr²⁺, respectively. Only Ca²⁺ resulted in a marked increase in the melting temperature (T_m) of the enzyme by 12-21 °C, while Ba²⁺, Mn²⁺, Sr²⁺ or Cu²⁺ resulted in a modest (2-3.5 °C) increase in T_m. In the presence of 1-5 mM Ca²⁺, the optimum temperature of the phytase activity was increased from 40 °C to 70 °C, while optimum pH of the enzyme shifted by 0.4-1 pH unit towards the acidic region.     

Characterisation of the Egeria densa Planch. leaf symplast

The hydrophyllic dyes fluorescein glutamic acid, fluorescein glutamylglutamic acid (F(Glu)₂), fluorescein hexaglycine, fluorescein leucyldiglutamyl-leucine and 6-carboxyfluorescein are unable to pass the plasmalemma in leaves of E. densa. However, when injected into single cells the dye conjugates of molecular weight 665 dalton or less move freely from cell-to-cell. This intercellular movement presumably occurs via the plant symplast. Movement of F(Glu)₂ from the injected cell occurs with greatly reduced frequency when Ca²⁺, Mg²⁺ or Sr²⁺ are injected into the cell immediately prior to the dye. The fraction of dye injections leading to movement declines with increasing group II ion concentration in the electrode tip, up to 10 mM. Sodium and K ions do not affect dye movement. When dye injection is delayed 30 min after Ca²⁺ injection, dye movement is no longer inhibited. Thus the cells recover from the Ca²⁺ injection, indicating that the ion does not cause major cell damage. Recovery from Mg²⁺ injection is not complete within 60 min. Treatment of leaves with chemicals expected to raise the concentration of free intracellular group II ions, notably the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, the inhibitor of mitochondrial Ca²⁺ uptake trifluralin, or the ionophore A23187 also inhibits dye movement, while the calmodulin inhibitor trifluoperazine does not. Cytoplasmic streaming is inhibited by Ca²⁺ or Mg²⁺ injection and by the metabolic inhibitors. However when streaming is stopped by cytochalasin B, dye movement is not inhibited. Hence steaming is not necessary for dye movement. Thus the cytoplasmic concentration of free group II ions may directly regulate the permeability of the plant symplast.     

Study of photophysical properties of different metal complexes of Alq3

Different metal complexes of Alq₃ (K⁺, Sr²⁺, Ca²⁺ and Mg²⁺) were synthesized by the precipitation method. The characterization of photoluminescence showed that presence of Mg²⁺ ion enhances photoluminescence of Alq₃ phosphor. The emission spectra are observed at 560 nm when excited at a wavelength of 440 nm. The phosphor is excited at a longer wavelength in the blue region of small energy, so that it could be used as lamp phosphor. It is observed that the prepared phosphor AlMgq₅ is more suitable than Alq_3. The ionic radii of K⁺, Sr²⁺, Ca²⁺ and Mg²⁺ ions are in decreasing order. Therefore, the remarkable properties of AlMgq₅ could be considered as promising materials as opto‐ or optoelectronic materials. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of the active site model for calcium-containing quinoprotein alcohol dehydrogenases

A new PQQ model compound [dimethyl 7-(1,4,7,10-tetraoxa-13-azacyclopentadec-13-yl)carbonyl-4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,9-dicarboxylate, 1], in which a 1-aza-15-crown-5 group is attached through an amide linkage at the 7-position, has been synthesized in order to develop an efficient model system of calcium-containing quinoprotein alcohol dehydrogenases. It has been found that Ca²⁺ binds to the quinone most strongly among the alkaline earth metal ions examined (Ca²⁺+>Sr²⁺+≫Ba²⁺+≫Mg²⁺) and the binding constant (K_M) for Ca²⁺ is as large as 2.1×10⁵ M⁻¹. Formation of the C-5 hemiacetal derivatives with ethanol is also investigated spectrophotometrically to show that the alcohol-addition to the quinone is enhanced in the presence of the metal ions. In this case, Ca²⁺ and Sr²⁺ show a similar efficiency that is several times larger than that of Ba²⁺. Addition of a strong base such as DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) into a MeCN solution containing the metal ion complex of 1 and ethanol leads to redox reactions to give the Ca²⁺ complex of 1H₂ (quinol form) and acetaldehyde. Kinetic studies on the redox reactions have been performed to gain insight into the mechanism of the alcohol-oxidation reaction catalyzed by the metal complexes of coenzyme PQQ.     

Effect of metal ion substitutions in anticoagulation factor I from the venom of Agkistrodon acutus on the binding of activated coagulation factor X and on structural stability

Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X (FXa)-binding protein that binds in a Ca²⁺-dependent fashion with marked anticoagulant activity. The thermodynamics of the binding of alkaline earth metal ions to ACF I and the effects of alkaline earth metal ions on the guanidine hydrochloride (GdnHCl)-induced unfolding of ACF I and the binding of ACF I to FXa were studied by isothermal titration calorimetry, fluorescence, circular dichroism, and surface plasmon resonance, respectively. The results indicate that the ionic radii of the cations occupying Ca²⁺-binding sites in ACF I crucially affect the binding affinity of ACF I for alkaline earth metal ions as well as the structural stability of ACF I against GdnHCl denaturation. Sr²⁺ and Ba²⁺, with ionic radii larger than the ionic radius of Ca²⁺, can bind to Ca²⁺-free ACF I (apo-ACF I), while Mg²⁺, with an ionic radius smaller than that of Ca²⁺, shows significantly low affinity for the binding to apo-ACF I. All bindings of Ca²⁺, Sr²⁺, and Ba²⁺ ions in two sites of ACF I are mainly enthalpy-driven and the entropy is unfavorable for them. Sr²⁺-stabilized ACF I exhibits slightly lower resistance to GdnHCl denaturation than Ca²⁺–ACF I, while Ba²⁺-stabilized ACF I exhibits much lower resistance to GdnHCl denaturation than Ca²⁺–ACF I. Mg²⁺ and Sr²⁺, with ionic radii close to that of Ca²⁺, can bind to FXa and therefore also induce the binding of ACF I to FXa, whereas Ba²⁺, with a much larger ionic radius than Ca²⁺, cannot support the binding of ACF I with FXa. Our observations suggest that bindings of Ca²⁺, Sr²⁺, and Ba²⁺ ions in two sites of ACF I increase the structural stability of ACF I, but these bindings are not essential for the binding of ACF I with FXa, and that the binding of Mg²⁺, Ca²⁺, and Sr²⁺ ions to FXa may be essential for the recognition between FXa and ACF I.     

Ca2+ and Mg2+ ions counteract the reduction by fosetyl-Al (aluminum tris[ethyl phosphonate]) of peroxidase activity from suspension-cultured grapevine cells

Grapevine (Vitis vinifera cv. Monastrell) cell suspension cultures were treated with 1.5 mM fosetyl-Al, a frequently used systemic fungicide for grapevine diseases caused by oomycetes. These cells showed a reduction in the level of peroxidase activity secreted into the culture media when compared to non-treated cells, the effect being mainly related to a decrease in the level of the basic B₁ peroxidase isozyme. The effect of fosetyl-Al on peroxidase was analogous to that observed with the Ca²⁺-channel blockers Co²⁺, Cd²⁺ and La³⁺, and was counteracted by Ca²⁺ ions, but was not reversed when the Ca²⁺-ionophore A23187 was added to the culture media. Moreover, the effect of fosetyl-Al on peroxidase activity and peroxidase isozymes was also partially reversed by Mg²⁺ ions but not by Sr²⁺, and was accentuated by Ba²⁺ ions. These results suggested that Ca²⁺ and Mg²⁺ ions specifically overcome the inhibitory effect of fosetyl-Al on peroxidase. In this context, an apoplastic Ca²⁺/Mg²⁺-displacement hypothesis is proposed for the mechanism of action of fosetyl-Al on peroxidase from grapevine cells.     

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.     

The interaction of Ca2+ with human factor IX

The binding isotherms of Ca²⁺ and Sr²⁺ to human blood coagulation Factor IX have been obtained at 25 °C and pH 7.4. In the case of both cations, a Scatchard plot of the data reveals that a single class of binding sites exist. For Ca²⁺, a total of 16.0 ± 1.0 sites, of K_D 7.3 ± 0.2 × 10^?4m, are present on human Factor IX. Similar analysis of the Sr²⁺ data indicates that Factor IX contains 11.0 ± 1.0 binding sites, with a K_D of 1.9 ± 0.1 × 10^?3m. Both Sr²⁺ and Mn²⁺ effectively displace Ca²⁺ from human Factor IX; whereas Mg²⁺ is considerably less potent in this regard. Conversely, Ca²⁺ is capable of nearly complete displacement of Sr²⁺ from its binding sites on human Factor IX. The activation of human Factor IX, by human Factor XIa, shows a complex dependence on the Ca²⁺ concentration. Sr²⁺ can substitute for Ca²⁺ in this activation process. Mn²⁺ cannot, in itself, substitute for Ca²⁺ in activation of Factor IX, but does significantly enhance the activation of Factor IX by Factor XIa at suboptimal levels of Ca²⁺. The rate of activation of human Factor IX by the coagulant protein of Russell's viper venom also shows a dependence on the presence of divalent cations. Here, however, a rigid specificity is not noted, since Ca²⁺, Sr²⁺, and Mn²⁺ all allow activation to proceed equally well.     

ATP and Ca2+ binding by the Ca2+ pump protein of sarcoplasmic reticulum

The binding of ATP and Ca²⁺ by the Ca²⁺ pump protein of sarcoplasmic reticulum from rabbit skeletal muscle has been studied and correlated with the formation of a phoshorylated intermediate. The Ca²⁺ pump protein has been found to contain one specific ATP and two specific Ca²⁺ binding sites per phosphorylation site. ATP binding is dependent on Mg²⁺ and is severely decreased when a phosphorylated intermediate is formed by the addition of Ca²⁺. In the presence of Mg²⁺ and the absence of Ca²⁺, ATP and ADP bind completely to the membrane. Pre-incubation with N-ethylmaleimide results in inhibition of ATP binding and decrease of Ca²⁺ binding. In the absence of ATP, Ca²⁺ binding is noncooperative at pH 6–7 and negatively cooperative at pH 8. Mg²⁺, Sr²⁺ and La³⁺, in that order, decrease Ca²⁺ binding by the Ca²⁺ pump protein. The affinity of the Ca²⁺ pump protein for both ATP and Ca²⁺ increases when the pH is raised from 6 to 8. At the infection point (pH ≈ 7.3) the binding constants of the Ca²⁺ pump protein-MgATP^2? and Ca²⁺ pump protein-calcium complexes are approx. 0.25 and 0.5 μM^?1, respectively. The unphosphorylated Ca²⁺ pump protein does not contain a Mg²⁺ binding site with an affinity comparable to those of the ATP and Ca²⁺ binding sites.The affinity of the Ca²⁺ pump protein for Ca²⁺ is not appreciably changed by the addition of ATP. The ratio of phosphorylated intermediate formed to bound Ca²⁺ is close to 2 over a 5-fold range of phosphoenzyme concentration. The equilibrium constant for phosphoenzyme formation is less than one at saturating levels of Ca²⁺. The phosphoenzyme is thus a “high-energy” intermediate, whose energy may then be used for the translocation of the two Ca²⁺.A reaction scheme is discussed showing that phosphorylation of sarcoplasmic reticulum proceeds via an enzyme-Ca₂²⁺-MgATP^2? complex. This complex is then converted to a phosphoenzyme intermediate which binds two Ca²⁺ and probably Mg²⁺.     

Contribution of carbohydrates to the cation-exchange selectivity of aquatic humus from peat-bog water

A sample of soluble humic acid from peat-bog water was a glycoconjugate containing 46% of a glycuronoglycan moiety and 54% of a dark-brown chromophore. These accounted for 37% and 63%, respectively, of the titratable acidity of the polymer. Cation-exchange capacities, and cationic selectivity coefficients relative to magnesium ions (K_Mg^Me), were measured on the humic acid for Pb²⁺, Cu²⁺, Zn²⁺, Ba²⁺, Ca²⁺, and Sr²⁺, and compared with those of extractive-free Sphagnum and other mosses, their chlorite holocelluloses, and two soluble fragments of Sphagnum holocellulose, prepared by acidic and alkaline degradation, respectively. The humic acid showed considerably higher K_Mg^Me values than most of the control materials, the enhancement being especially marked for Pb²⁺, Cu²⁺, and Ca²⁺. Scatchard plots showed that both parts of the glycoconjugate contributed to its selectivity, and that the selectivity of the carbohydrate part was greater in the humic acid than in the holocellulose or its soluble fragments. The results are explained by assuming that there are enhanced possibilities for cross-linking in the colloidal humic-acid complexes.     

Properties of Ca2+ uptake and release by Golgi membrane vesicles from rat intestine

A previous study of energy-independent in vitro Ca²⁺ uptake by rat intestinal epithelial membrane vesicles demonstrated that uptake by Golgi membrane vesicles was greater than that by microvillus or lateral-basal membrane vesicles, was markedly decreased in vitamin D-deficient rats, and responded specifically to 1,25-(OH)₂D₃ repletion (R. A. Freedman, M. M. Weiser, and K. J. Isselbacher, 1977, Proc. Nat. Acad. Sci. USA74, 3612–3616; J. A. MacLaughlin, M. M. Weiser, and R. A. Freedman, 1980, Gastroenterology78, 325–332). In the present study, properties of Ca²⁺ uptake and release by intestinal Golgi membrane vesicles have been investigated. The initial rate of uptake was found to be saturable, suggesting carrier-mediated uptake. Uptake was markedly inhibited by Mg²⁺ and Sr²⁺, but not by Na⁺ or K⁺. Lowering the external [H⁺] or raising the internal [H⁺] resulted in enhancement of the initial rate of uptake; the intial rate was found to correlate with the internal-to-external [H⁺] gradient. The initial rate of uptake could be enhanced by preloading the vesicles with MgCl₂ or SrCl₂ but not CaCl₂, NaCl, or KCl. Vesicles preloaded with K₂SO₄ failed to show enhanced uptake in the presence of valinomycin, suggesting that enhancement in uptake by vesicles preloaded with MgCl₂ was not due to transmembrane potentials. The internal volume of the Golgi membrane vesicles was determined and found to be 9 μl/mg protein; this volume could accomodate less than 1% of the Ca²⁺ uptake maintained at equilibrium. Therefore, the remainder of the Ca²⁺ taken up was presumably bound to the Golgi membranes. A dissociation constant of 3.8 × 10^?6m was found for this binding. The bound Ca²⁺ could be rapidly released by external Mg²⁺ or Sr²⁺, but not Ca²⁺, Na⁺, or K⁺. Release of bound Ca²⁺ could also be induced by raising the [H⁺] of the external medium. Failure of external Ca²⁺ to release bound Ca²⁺ suggested that the release induced by external Mg²⁺, Sr²⁺, or H⁺ was not due to competitive displacement of Ca²⁺ from its binding sites. These results indicated that Ca²⁺ uptake by intestinal Golgi membrane vesicles consists of carrier-mediated transport followed by binding of Ca²⁺ to the vesicle. The effects of H⁺, Mg²⁺, and Sr²⁺ on Ca²⁺ uptake and release suggest the existence of cation countertransport in the Golgi membrane vesicles.     

Fluxes of Ca2+, Sr2+ and Mg2+ in synaptosomes.

Synaptosomes isolated from sheep brain cortex accumulate Ca²⁺, Sr²⁺ and Mg²⁺ when incubated in isosmotic sucrose media containing 5 mM of either of these cations. The maximal levels of cations retained per mg of protein are 100 nmol of Ca²⁺, 85 nmol of Mg²⁺ and 80 nmol of Sr²⁺. The loss of Ca²⁺ or Sr²⁺ from the preloaded synaptosomes is increased by monovalent cations in the following order: Na⁺> K⁺ > Li⁺> choline, whereas for the loss of Mg²⁺ this order is different: K⁺ > Na⁺ > Li ～ choline. The efflux of Ca²⁺ or Sr²⁺ induced by monovalent cations decreases as the temperature is lowered and it is nearly abolished at 0°C, whereas the efflux of Mg²⁺ is much less influenced by temperature. The results suggest that the mechanism of exchange of Ca²⁺ for Na⁺ in synaptosomes operates similarly for Sr²⁺, but not for Mg²⁺.     

Ionophorous properties of the 20,000-dalton fragment of (Ca2++Mg2+)-ATPase in phosphatidylcholine: Cholesterol membranes

Summary The purified 20,000-dalton fragment of sarcoplasmic reticulum (Ca²⁺+Mg²⁺)-ATPase has been shown by us (A.E. Shamoo, T.E. Ryan, P.S. Stewart, D.H. MacLennan, 1976. J. Biol. Chem.251:4147) to have Ca²⁺-selective ionophoric activity. The Ca²⁺-ionophoric fragment has been purified by either SDS-column chromatography or SDS-preparative gel electrophoresis. The Ca²⁺-ionophoric fragment has been subjected to prolonged dialysis to insure the removal of bound SDS from the fragment. The selectivity sequence of this fragment in black lipid membranes (BLM) formed from either oxidized cholesterol or phosphatidylcholine/cholesterol is the same,P _Ba>P _Ca>P _Sr>P _Mg>P _Mn. This selectivity sequence is the same as that for the intact (Ca²⁺ +Mg²⁺)-ATPase. Treatment of the fragment with cholate to absolutely insure the removal of bound SDS resulted in the fragment having a selectivity sequence as above except thatP _Mn>P _Mg. This and other data indicate that the 20,000-dalton fragment is the site containing the Ca²⁺-ionophoric activity of the (Ca²⁺+Mg²⁺)-ATPase.     

Evidence for solvent-induced conformational changes of the soluble Dunaliella chloroplast coupling factor 1 (CF1)

The ATPase activity of the chloroplast coupling factor 1 (CF₁) isolated from the green alga Dunaliella is completely latent. A brief heat treatment irreversibly induces a Ca²⁺ -dependent activity. The Ca²⁺ dependent ATPase activity can be reversibly inhibited by ethanol, which changes the divalent cation dependency from Ca²⁺ to Mg²⁺. Both the Ca²⁺ -dependent and Mg²⁺ -dependent ATPase activities of heat-treated Dunaliella CF₁ are inhibited by monospecific antisera directed against Chlamydomonas reinhardi CF₁. However, when assayed under identical conditions, the Ca²⁺ -dependent ATPase activity is significantly more sensitive to inhibition by the antisera than is the Mg²⁺ -dependent activity. These data are interpreted as indicating that soluble Dunaliella CF₁ can exist in a variety of conformations, at least one of which catalyzes a Ca²⁺ -dependent ATPase and two or more of which catalyze an Mg²⁺ -dependent ATPase.     

Flash-induced consumption of molecular oxygen on the donor side of photosystem II in Mn-depleted subchloroplast membrane fragments: specific effects of manganese and calcium ions

It has been shown that removal of manganese from the water-oxidizing complex (WOC) of photosystem II (PSII) leads to flash-induced oxygen consumption (FIOC) which is activated by low concentration of Mn²⁺ (Yanykin et al., Biochim Biophys Acta 1797:516–523, 2010). In the present work, we examined the effect of transition and non-transition divalent metal ions on FIOC in Mn-depleted PSII (apo-WOC-PSII) preparations. It was shown that only Mn²⁺ ions are able to activate FIOC while other transition metal ions (Fe²⁺, V²⁺ and Cr²⁺) capable of electron donation to the apo-WOC-PSII suppressed the photoconsumption of O₂. Co²⁺ ions with a high redox potential (E ⁰ for Co²⁺/Co³⁺ is 1.8 V) showed no effect. Non-transition metal ions Ca²⁺ by Mg²⁺ did not stimulate FIOC. However, Ca²⁺ (in contrast to Mg²⁺) showed an additional activation effect in the presence of exogenic Mn²⁺. The Ca²⁺ effect depended on the concentration of both Mn²⁺ and Ca²⁺. The Ca effect was only observed when: (1) the activation of FIOC induced by Mn²⁺ did not reach its maximum, (2) the concentration of Ca²⁺ did not exceed 40 μM; at higher concentrations Ca²⁺ inhibited the Mn²⁺-activated O₂ photoconsumption. Replacement of Ca²⁺ by Mg²⁺ led to a suppression of Mn²⁺-activated O₂ photoconsumption; while, addition of Ca²⁺ resulted in elimination of the Mg²⁺ inhibitory effect and activation of FIOC. Thus, only Mn²⁺ and Ca²⁺ (which are constituents of the WOC) have specific effects of activation of FIOC in apo-WOC-PSII preparations. Possible reactions involving Mn²⁺ and Ca²⁺ which could lead to the activation of FIOC in the apo-WOC-PSII are discussed.     

Ca2+-stimulated,Mg2+-independent ATP hydrolysis and the high affinity Ca2+-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg²⁺-dependency of Ca²⁺-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg²⁺- and Ca²⁺-buffering ligands. ATP hydrolysis is strongly stimulated by Mg²⁺ with a K_m of 13 μ M in the absence or presence of 1 μ M free Ca²⁺. At free Mg²⁺ concentrations of 1 μ M and lower, ATP hydrolysis is Mg²⁺ -independent, but is strongly stimulated by submicromolar Ca²⁺ concentrations K_m  0.25 μM, V_max  24 μmol P_i/h per mg protein). The Ca²⁺-stimulated ATP hydrolysis strongly decreases at higher Mg²⁺ concentrations. The Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg²⁺ concentrations calmodulin and trifluoperazine affect the high affinity Ca²⁺-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca²⁺-pumping in renal basolaterals and on Ca²⁺-ATPase in erythrocyte ghosts suggest that the Ca²⁺-pumping enzyme requires Mg²⁺. In contrast, a role of the Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis in active Ca²⁺ transport across basolateral membranes is rather unlikely.