Effect of calcium and magnesium ions on different stages of genetic transformation in Bacillus subtilis]

Optimal concentrations of magnesium and calcium ions under their combined effect on genetic transformation in Bacillus suttilis are 2 - 10(-2) M and 4 - 10(-2) M respectively. The same concentrations are optimal under the effect of each cation clone. Magnesium ions are efficient during irreversible DNA binding. In the presence of magnesium ions calcium ions stimulate more late stages of transformation. The greatest efficiency of transformation is shown in consecutive effect of magnesium ions at early stages of transformation and of calcium ions at late transformation stages. This suggests that magnesium and calcium ions stimulate the activity of nuclease, taking part at early and late transformation stages.     

Calcium-promoted DNA cleavage by eukaryotic topoisomerase II: trapping the covalent enzyme-DNA complex in an active form

The effects of calcium ions on interactions between Drosophila melanogaster topoisomerase II and DNA were assessed. Although the divalent cation could not support DNA strand passage, it was able to promote high levels of enzyme-mediated DNA cleavage. Moreover, sites of cleavage on plasmid pBR322 generated in calcium-promoted reactions were similar to those obtained in the presence of magnesium. When calcium-containing enzyme-DNA mixtures were treated with ethylenediaminetetraacetic acid, cleaved nucleic acids could be generated in the absence of sodium dodecyl sulfate (SDS) or other denaturing detergents. The product of this SDS-independent calcium-promoted reaction was a covalent topoisomerase II-DNA complex. Enzyme molecules trapped in such complexes were found to be kinetically competent. Therefore, calcium should be a valuable tool for studying the enzymology of topoisomerase II mediated DNA cleavage.     

Interaction of mithramycin with metal ions and DNA

The interaction of mithramycin with metal ions has been studied by absorbance and fluorescence spectroscopy. Magnesium shifts the drug absorbance spectrum to longer wavelengths and displays a weak binding constant (Kd = 1mM); no interaction with calcium was detected. The drug requires magnesium for binding to DNA and this is characterised by small additional hypochromic and bathochromic changes. Mithramycin does not bind to DNA in the presence of calcium. With 10mM magnesium the drug binds to DNA with an association constant of 9.2 x 10(4) M-1. The inability of calcium to substitute for magnesium has been confirmed by 'footprinting' experiments using both DNase I and hydroxyl radicals.     

Deficiency of calcium and magnesium induces apoptosis via scavenger receptor BI

AimsCells undergo apoptosis in stressed status such as in intracellular calcium overload or extracellular calcium/magnesium deficiency. The mechanisms of how deficiency of the divalent metal ions induces apoptosis remain to be defined. Scavenger receptor BI (SR-BI) is a high density lipoprotein (HDL) receptor. Recent studies demonstrated that SR-BI is a stress response molecule which induces apoptosis upon serum deprivation. In this study, we assessed our hypothesis that the deficiency of calcium/magnesium induces apoptosis via SR-BI apoptotic pathway.Main methodsWe employed CHO cell lines expressing vector and SR-BI to test the effect of SR-BI on apoptosis induced by deficiency of calcium, magnesium and zinc in culture medium. The regain of different metal ions in deficient medium was also performed, respectively. Cell death was detected by morphological changes and quantified by LDH cytotoxicity assay. Apoptosis was also assessed by DNA ladder assay and DNA condensation assay. The SR-BIC323G mutant cells which lack the apoptotic activity of SR-BI were employed to verify the SR-BI-dependent effect on calcium/magnesium induced apoptosis.Key findingsThe deficiency of calcium/magnesium induced cell apoptosis in CHO-SR-BI cells, but not in CHO-vector cells. Moreover, no apoptotic cell death was observed in SR-BIC323G mutant cells, indicating that the deficiency of divalent metal ions induces apoptosis in a SR-BI-dependent manner. Furthermore, the restoration of calcium or magnesium, but not zinc, protected CHO-SR-BI cells from apoptotic cell death, in a dose-dependent fashion.SignificanceThese findings extend our understanding about how calcium and magnesium deficiency induces apoptosis.     

Why do divalent metal ions either promote or inhibit enzymatic reactions? The case of BamHI restriction endonuclease from combined quantum-classical simulations

Divalent metal ions are essential to many enzymatic reactions involving nucleic acids, but their critical and specific role still needs to be uncovered. Restriction endonucleases are a prominent group of such metal-requiring enzymes. Large scale accurate simulations of Mg- and Ca-BamHI elucidate the mechanism of the catalytic reaction leading to DNA cleavage and show that it involves the concerted action of two metal ions and water molecules. It is also established that what is decisive for the dramatically different behavior of magnesium (a cocatalyst) and calcium (an inhibitor) are kinetic factors and not the properties of the prereactive states of the enzymes. A new perspective is opened for the understanding of the functional role of metal ions in biological processes.     

Crystal structure of a c-kit promoter quadruplex reveals the structural role of metal ions and water molecules in maintaining loop conformation

We report here the 1.62 Å crystal structure of an intramolecular quadruplex DNA formed from a sequence in the promoter region of the c-kit gene. This is the first reported crystal structure of a promoter quadruplex and the first observation of localized magnesium ions in a quadruplex structure. The structure reveals that potassium and magnesium ions have an unexpected yet significant structural role in stabilizing particular quadruplex loops and grooves that is distinct from but in addition to the role of potassium ions in the ion channel at the centre of all quadruplex structures. The analysis also shows how ions cluster together with structured water molecules to stabilize the quadruplex arrangement. This particular quadruplex has been previously studied by NMR methods, and the present X-ray structure is in accord with the earlier topology assignment. However, as well as the observations of potassium and magnesium ions, the crystal structure has revealed a highly significant difference in the dimensions of the large cleft in the structure, which is a plausible target for small molecules. This difference can be understood by the stabilizing role of structured water networks.     

The role of hydrated divalent metal ions in the bridging of two anionic groups. An ab initio quantum chemical and molecular mechanics study of dimethyl phosphate and formate bridged by calcium and magnesium ions

Ab initio quantum chemical (Gaussian82) and molecular mechanics (AMBER2.0) computational techniques are employed to investigate the interaction of two anions (formate an dimethylphosphate) and a central divalent metal cation (magnesium or calcium). These systems are models for the essential GDP binding unit of the G-proteins (e.g., EF-Tu or the ras oncogene proteins) and for protein/phospholipid interactions, both of which are mediated by divalent metal cations. Various levels of hydration are utilized to examine coordination of differences between magnesium and calcium ions. Two different orientations of formate and dimethyl phosphate in direct ion contact with a magnesium ion and two waters of hydration were energy minimized with both quantum and molecular mechanics techniques. The structures and energy differences between the two orientations determined by either of the computational techniques are similar. Magnesium ion has a strong propensity to assume six coordination whereas calcium ion preferentially assumes a coordination greater than six. Likewise, water molecules attached to magnesium ion are held more rigidly than those of calcium ion, thus calcium ion is more accommodating in the exchange of water for negative ligands.     

On the importance of calcium and magnesium ions in yeast sporulation

Irrespective of the nutritional conditions, the sporulation frequency of wild and industrially used yeasts on agar or agarose plates has been found to vary from one experiment to another. An analysis of agar- and agarose-extracts by ion-exchange column chromatography proved that the amount of calcium and/or magnesium ions contained in the agar was a factor in the fluctuation of sporulation frequency. Furthermore, these two cations enhanced the formation of four-spored asci. When calcium or magnesium ions were added to a nutrition-deprived medium solidified with agarose containing no detectable calcium and magnesium ions, wild and industrially used sake yeasts efficiently sporulated with a frequency of 10–40%. A strictly controlled sporulation condition suitable for the analysis of meiosis and sporulation of yeast cells was constructed by using calcium and/or magnesium ions and highly purified agarose.     

Microcalorimetric study of heat denaturation of DNA from calf thymus DNA in the absence of magnesium ions

Thermal denaturation was studied for a wide range of magnesium ions concentrations and salt concentration 0.15 M NaCl. It was shown that thermal stability of DNA increases at low Mg/2P ratios and decreases at high concentrations of magnesium ions. Up to Mg/2P = 10 DNA denaturation is an equilibrium process. With an increase in magnesium ions concentrations the enthalpy of DNA denaturation reaches the maximum at Mg/2P = 10 (50 kJ/mole base pairs). DNA aggregation and appearance of a new heat absorption peak is observed in the high temperature region at Mg/2P = 10. At this region of magnesium ions concentrations DNA denaturation process is non-equilibrium.     

Three metal ions participate in the reaction catalyzed by T5 flap endonuclease

Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the rapid hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are widely proposed to catalyze phosphate diester hydrolysis using a "two-metal-ion mechanism." Yet, analyses of flap endonuclease (FEN) family members, which occur in all domains of life and act in DNA replication and repair, exemplify controversies regarding the classical two-metal-ion mechanism for phosphate diester hydrolysis. Whereas substrate-free structures of FENs identify two active site metal ions, their typical separation of > 4 A appears incompatible with this mechanism. To clarify the roles played by FEN metal ions, we report here a detailed evaluation of the magnesium ion response of T5FEN. Kinetic investigations reveal that overall the T5FEN-catalyzed reaction requires at least three magnesium ions, implying that an additional metal ion is bound. The presence of at least two ions bound with differing affinity is required to catalyze phosphate diester hydrolysis. Analysis of the inhibition of reactions by calcium ions is consistent with a requirement for two viable cofactors (Mg2+ or Mn2+). The apparent substrate association constant is maximized by binding two magnesium ions. This may reflect a metal-dependent unpairing of duplex substrate required to position the scissile phosphate in contact with metal ion(s). The combined results suggest that T5FEN primarily uses a two-metal-ion mechanism for chemical catalysis, but that its overall metallobiochemistry is more complex and requires three ions.     

The Role of Hydrated Divalent Metal Ions in the Bridging of Two Anionic Groups. An ab initio Quantum Chemical and Molecular Mechanics Study of Dimethyl Phosphate and Formate Bridged by Calcium and Magnesium Ions

Abstract

Ab initio quantum chemical (Gaussian82) and molecular mechanics (AMBER2.0) computational techniques are employed to investigate the interaction of twoanions (formate and dimethyl phosphate) and a central divalent metal cation (magnesium or calcium). These systems are models for the essential GDP binding unit of the G-proteins (e.g., EF-Tu or the ras oncogene proteins) and for protein/phospholipid interactions, both of which are mediated by divalent metal cations. Various levels of hydration are utilized to examine coordination of differences between magnesium and calcium ions. Two different orientations of formate and dimethyl phosphate in direct ion contact with a magnesium ion and two waters of hydration were energy minimized with both quantum and molecular mechanics techniques. The structures and energy differences between the two orientations determined by either of the computational techniques are similar. Magnesium ion has a strong propensity to assume six coordination whereas calcium ion preferentially assumes a coordination greater than six. Likewise, water molecules attached to magnesium ion are held more rigidly than those to calcium ion, thus calcium ion is more accommodating in the exchange of water for negative ligands.     

Influence of copper(II) and magnesium(II) ions on the ciprofloxacin binding to DNA

The influence of magnesium(II) and copper(II) ions on the binding of ciprofloxacin to double stranded calf thymus DNA was studied by fluorescence emission spectroscopy, ultraviolet- and circular dichroism (CD) spectroscopy. The interaction of ciprofloxacin and copper(II) ions was followed by strong fluorescence quenching which was almost unaffected by the presence of DNA. On the other hand, only a slight decrease in fluorescence emission intensity, which was enhanced in the presence of DNA, was observed for ciprofloxacin interaction with magnesium(II) ions. Furthermore, magnesium(II) ions increase the thermal stability of the DNA, while, in the presence of ciprofloxacin, the degree of stabilisation is smaller. In contrast, copper(II) ions destabilise double helical DNA to heat, while ciprofloxacin slightly affects only the second transition of the biphasic melting curve of calf thymus DNA. Magnesium(II) ions at 25 degrees C induce conformational transitions of DNA at concentrations of 1.5 mM and 2.5 M, as monitored by CD. On the other hand copper(II) ions induce only one conformational transition, at a concentration of 12.7 microM. At higher concentrations of copper(II) ions (c>700 microM) DNA starts to precipitate. Significant changes in the CD spectra of DNA were observed after addition of ciprofloxacin to a solution containing DNA and copper(II) ions, but not to DNA and magnesium(II) ions. Based on our spectroscopic results, we propose that copper(II) ions are not directly involved into ciprofloxacin binding to DNA via phosphate groups as it has been suggested for magnesium(II) ions.     

Quantitative DNA slot blot analysis: inhibition of DNA binding to membranes by magnesium ions.

Titers of wild-type and recombinant adeno-associated viruses are routinely determined by DNA slot blot analysis. The binding of viral DNA to nylon membranes was found to be inhibited by magnesium ions, which are critical components of the DNase I digestion carried out prior to slot blot analysis. Mg2+ions also interfered with the adsorption of plasmid DNA to nylon and nitrocellulose membranes. These observations yield practical insights into the poorly understood mechanisms by which DNA molecules are retained on solid supports.     

Effect of calcium ions on creatine kinase systems of myocardial cells

The kinetics of the creatine phosphokinase reaction catalyzed by different creatine phosphokinase (CPK) isoenzymes from the heart and the mechanism of the regulatory action of calcium ions on this reaction have been studied. It has been shown that the kinetic parameters of the reaction in the presence of calcium are similar to those in the presence of magnesium with the exception of the maximal rate of the reverse reaction, which is about three times lower in the presence of calcium. Calcium ions are able to exert a significant regulatory action on the CPK reaction. Simultaneous regulation of the CPK reaction by calcium and magnesium ions can be quantitatively described by a kinetic model, which takes into account the formation of complexes of adenine nucleotides with calcium and magnesium. The mechanism of the CPK reaction regulation by metal ions involves changes in concentrations of the metal -- adenine nucleotide compexes. The regulatory action of calcium on the creatine kinase reaction is the same for all CPK isoenzymes studied.     

25Mg-nmr investigations of the magnesium ion–DNA interaction

²⁵Mg-nmr data are reported that address the nature of the magnesium ion–DNA interaction. It is found that competitor ions such as calcium, mercury, zinc, and cobalt ions are not effective in competing for all of the magnesium ion–DNA interaction that is reported by the ²⁵Mg-nmr spectrum. The temperature dependence of the ²⁵Mg-nmr spectrum in DNA solution studied at high concentrations of competitor ion indicates that the chemical-exchange lifetime of the magnesium ions at DNA binding sites makes a major contribution to the ²⁵Mg-nmr line width. However, the activation parameters are not consistent with the temperature dependence of either transport properties or chemical exchange with phosphate groups alone, but are consistent with a sum of at least two processes that provide opposing contributions to the ²⁵Mg-nmr relaxation. It is also shown that the non-Lorentzian character of the ²⁵Mg-nmr line previously reported is consistent with the effect of an incompletely averaged static nuclear electric quadrupole interaction and/or an exchange process that is slow with respect to the magnitude of this interaction. Because the concentrations employed in these experiments are high, the present data do not provide a direct or critical test of the electrostatic theories of ion–polyelectrolyte interaction. The present data do demonstrate, however, that such theories alone are insufficient as a basis for understanding the ²⁵Mg-nmr data.     

Influence of magnesium ions on helix-coil transition of DNA determined by modified differential scanning calorimeter

The thermal effect of magnesium ions on the helix–coil transition of DNA was studied calorimetrically by a modified differential scanning calorimeter (DSC). It was found that the transition temperature of DNA depends on both the DNA and magnesium ion concentrations. The dependence of the helix–coil transition of DNA on the mole ratio of magnesium ions to DNA(P) can be classified into two groups. When this mole ratio is less than 1, magnesium ions tend to stabilize the double-helix DNA, so that the transition temperature increases linearly and the heat of transition increases significantly with increasing mole ratio. When the mole ratio is more than 1, magnesium ions tend to destabilize the double-helix DNA, so that DNA precipitates when the temperature is raised above the transition temperature. In this case, both the transition temperature and the heat of transition decrease with increasing mole ratio.     

胞外镁离子对SD成年大鼠心肌细胞胞内钙信号的影响

目的：研究胞外不同浓度的镁离子对SD成年大鼠心肌细胞钙瞬变的影响。方法：采用激光共聚焦显微镜系统同步配合阈上电刺激探测心肌细胞钙瞬变。结果：胞外低浓度的镁离子（0 mmol /L,0.5 mmol /L; 正常镁离子浓度为1 mmol/L）可以升高钙瞬变的峰值（P〈0.05）,但不影响钙瞬变的持续时间（以钙瞬变的半高宽表示）（P〉0.05）;胞外高浓度的镁离子（2.5 mmol /L,5 mmol /L,10 mmol /L）均可抑制钙瞬变的峰值（P〈0.05）;其中5 mmol/L和10 mmol/L的胞外镁还能延长钙瞬变的持续时间（P〈0.05）。结论：正常情况下镁对心肌细胞钙瞬变有抑制作用。     

The minerals of milk

The salt of milk constitutes a small part of milk (8-9 g.L(-1)); this fraction contains calcium, magnesium, sodium and potassium for the main cations and inorganic phosphate, citrate and chloride for the main anions. In milk, these ions are more or less associated between themselves and with proteins. Depending on the type of ion, they are diffusible (cases of sodium, potassium and chloride) or partially associated with casein molecules (cases of calcium, magnesium, phosphate and citrate), to form large colloidal particles called casein micelles. Today, our knowledge and understanding concerning this fraction is relatively complete. In this review, the different models explaining (i) the nature and distribution of these minerals (especially calcium phosphate) in both fractions of milk and (ii) their behaviour in different physico-chemical conditions, are discussed.     

Cytokinins and magnesium ions may control the flow of metabolites and calcium ions through fungal cell membranes

Compounds that are known to display cytokinin activity in green plants function as allosteric regulators of metabolite and ion transport in fungal cells. These hormonal compounds stimulate a massive release of calcium bound to a glycoprotein localised on the membrane surface, and simultaneously, activate the transport of calcium into the cells. The energy-linked import of sugars, nucleosides, and amino acids is inhibited by the cytokinins. The activating effect of cytokinins is neutralised by magnesium ions. Calcium and metabolite import in the fungi studied appear to depend upon a delicate balance between the concentrations of cytokinins, calcium, and magnesium ions.