Sword bean variants and different pretreatments influence protein extraction and protein properties

Two variants of the sword bean (Canavalia gladiata), namely the white sword bean (WSB) and the red sword bean (RSB), are known. The MgCl₂ concentration-dependent canavalin solubility showed different behavior among the extracts from distinct beans prepared by distinct pretreatments. Pretreatment and bean selection are important factors for use in food chemical and biochemical experiments.     

Evidence that a light-accelerated abrupt change in membrane characteristics of bovine rod outer segment fragments takes place at acidic pH

Changes in the turbidity of suspensions of bovine rod outer segment fragments induced by rhodopsin bleaching were measured in the presence of various concentrations of divalent cations at acidic pH (4.7–5.4). Unlike the situation at neutral pH, the turbidity of the suspensions increased drastically by bleaching at acidic pH. It was found that the extent of turbidity change became maximum at a particular concentration of divalent cations (i.e., 5 mM CaCl₂, 5 mM MgCl₂, or 5 mM mixed divalent cations). However, the turbidity increment in the presence of 5 mM MgCl₂ was greatly enhanced by the addition of a minute amount of CaCl₂. These results evidently show that the membrane characteristic is abruptly changed by bleaching at acidic pH in particular. It is also suggested that there are two kinds of binding sites for Ca ions: one is a Ca²⁺ specific site, and the other is a nonspecific site to which Mg²⁺ can also bind.     

Modifications of the functioning of Photosystem II induced in the dark by alkaline pH in the presence of cations

Submission of chloroplasts to alkaline pH, in the range pH 7.5–9.5, leads to changes in their oxygen-evolving capacities. These changes are enhanced by the addition of divalent cations and also monovalent cations at high concentrations. (1) Dark incubation of chloroplasts at pH ? 9 gives rise to a time-dependent inactivation of electron transport from water to 2,6-dichlorophenolindophenol measured at neutral pH. The rate of inactivation is increased by adding cations. (2) The variable fluorescence is decreased with a dependence on incubation time and concentration of cations similar to that of the Hill reaction. Addition of the electron donor NH₂OH removes most of the fluorescence quenching, (3) EPR measurements indicate that the inactivations are accompanied by loss of Mn²⁺ and the appearance of signal II fast. (4) At lower pH (7.5) the oscillations of oxygen evolved per flash during a sequence of flashes show an increase in damping when 20 mM MgCl₂ is present instead of 100 mM KCI. These changes are not seen at pH 6. (5) None of these Mg²⁺-induced modifications are prevented by glutaraldehyde fixation. We conclude that the effects of alkaline pH and MgCl₂ do not involve major protein structural changes, and that both act on the manganese-containing protein of the oxygen-evolving site.     

Precipitation of sword bean proteins by heating and addition of magnesium chloride in a crude extract

Sword bean (Canavalia gladiata) seeds are a traditional food in Asian countries. In this study, we aimed to determine the optimal methods for the precipitation of sword bean proteins useful for the food development. The soaking time for sword beans was determined by comparing it with that for soybeans. Sword bean proteins were extracted from dried seeds in distilled water using novel methods. We found that most proteins could be precipitated by heating the extract at more than 90 °C. Interestingly, adding magnesium chloride to the extract at lower temperatures induced specific precipitation of a single protein with a molecular weight of approximately 48 kDa. The molecular weight and N-terminal sequence of the precipitated protein was identical to that of canavalin. These data suggested that canavalin was precipitated by the addition of magnesium chloride to the extract. Our results provide important insights into the production of processed foods from sword bean.     

Regulatory Effect of Magnesium Ions on Interaction of Prolactin and Somatotropin with Receptors on Granulosum Cells of the Cow Bos taurus

A comparative study of effects of Mg²⁺ ions on specific and competitive binding of the bovine somatotropic hormone (STH) and bovine prolactin (PL) with cells of cow granulosum is carried out. It is found that Mg²⁺ increases the level of specific binding of ¹²⁵I-PL with the cells at concentrations from 1 to 70 mM and decreases the level of specific binding of ¹²⁵I-STH at a concentration of 70 mM. Analysis of the data by Scatchard's method has shown that the decrease of the level of specific binding of ¹²⁵I-PL in the absence of MgCl₂ and ¹²⁵I-STH at a MgCl₂ concentration of 70 mM is caused mainly by a decrease of the number of active binding sites on the cells. Oppositely directed effects of the studied divalent cations on the capability of unlabelled STH (25 µg/ml) and PL (25 µg/ml) for cross-suppression of specific binding of ¹²⁵I-PL and ¹²⁵I-STH, respectively, with the granulosum cells have been revealed. At the same time, with increase of Mg²⁺ ion concentration, the degree of replacement of ¹²⁵I-STH and ¹²⁵I-PL with unlabelled STH and PL, respectively, did not change. The obtained results are considered in connection with electrostatic model of participation of the divalent cations in interaction of PL and STH with receptors on cells.     

Effect of surface potential on the intramembrane electrical field measured with carotenoid spectral shift in chromatophores from Rhodopseudomonas sphaeroides

Changes in the surface potential, the electrical potential difference between the membrane surface and the bulk aqueous phase were measured with the carotenoid spectral shift which indicates the change of electrical field in the membrane. Chromatophores were prepared from a non-sulfur purple bacterium, Rhodopseudomonas sphaeroides, in a low-salt buffer. Surface potential was changed by addition of salt or by pH jump as predicted by the Gouy-Chapman diffuse double layer theory.When a salt was added at neutral pH, the shift of carotenoid spectrum to shorter wavelength, corresponding to an increase in electrical potential at the outside surface, was observed. The salts of divalent cations (MgSO₄, MgCl₂, CaCl₂) were effective at concentrations lower than those of monovalent cation salts (NaCl, KCl, Na₂SO₄) by a factor of about 50. Among the salts of monoor divalent cation used, little ionic species-dependent difference was observed in the low-concentration range except that due to the valence of cations. The pH dependence of the salt-induced carotenoid change was explained in terms of the change in surface charge density, which was about 0 at pH 5–5.5 and had negative values at higher pH values. The dependence of the pH jump-induced absorbance change on the salt concentration was also consistent with the change in the charge density. The surface potential change by the salt addition, which was calibrated by H⁺ diffusion potential, was about 90 mV at the maximum. From the difference between the effective concentrations with salts of mono- and divalent cations at pH 7.8, the surface charge density of (?1.9 ± 0.5) · 10^?3 elementary charge per Ų, and the surface potential of about ?100 mV in the presence of about 0.1 mM divalent cation or 5 mM monovalent cation were calculated.     

Cation-permeable vacuolar ion channels in the moss Physcomitrella patens: a patch-clamp study

Patch-clamp studies carried out on the tonoplast of the moss Physcomitrella patens point to existence of two types of cation-selective ion channels: slowly activated (SV channels), and fast-activated potassium-selective channels. Slowly and instantaneously saturating currents were observed in the whole-vacuole recordings made in the symmetrical KCl concentration and in the presence of Ca²⁺ on both sides of the tonoplast. The reversal potential obtained at the KCl gradient (10 mM on the cytoplasmic side and 100 mM in the vacuole lumen) was close to the reversal potential for K⁺ (E _K), indicating K⁺ selectivity. Recordings in cytoplasm-out patches revealed two distinct channel populations differing in conductance: 91.6 ± 0.9 pS (n = 14) at ?80 mV and 44.7 ± 0.7 pS (n = 14) at +80 mV. When NaCl was used instead of KCl, clear slow vacuolar SV channel activity was observed both in whole-vacuole and cytoplasm-out membrane patches. There were no instantaneously saturating currents, which points to impermeability of fast-activated potassium channels to Na⁺ and K⁺ selectivity. In the symmetrical concentration of NaCl on both sides of the tonoplast, currents have been measured exclusively at positive voltages indicating Na⁺ influx to the vacuole. Recordings with different concentrations of cytoplasmic and vacuolar Ca²⁺ revealed that SV channel activity was regulated by both cytoplasmic and vacuolar calcium. While cytoplasmic Ca²⁺ activated SV channels, vacuolar Ca²⁺ inhibited their activity. Dependence of fast-activated potassium channels on the cytoplasmic Ca²⁺ was also determined. These channels were active even without Ca²⁺ (2 mM EGTA in the cytosol and the vacuole lumen), although their open probability significantly increased at 0.1 μM Ca²⁺ on the cytoplasmic side. Apart from monovalent cations (K⁺ and Na⁺), SV channels were permeable to divalent cations (Ca²⁺ and Mg²⁺). Both monovalent and divalent cations passed through the channels in the same direction—from the cytoplasm to the vacuole. The identity of the vacuolar ion channels in Physcomitrella and ion channels already characterised in different plants is discussed.     

Fatty Acid Synthesis in Leucoplasts Isolated from Developing Seeds of Brassica campestris

Fatty acid synthesis from Na (1-¹⁴C) acetate in leucoplasts isolated from developing seeds of Brassica compestris was found to be maximum when leucoplasts were supplied with 0.8 mM acetate, 20 mM NaHCO₃, 8 mM ATP, 8 mM MgCl₂, 4 mM MnCl₂, 0.6 mM CoA, 1 mM NADH, 1 mM NADPH and 0.2 M sorbitol and incubated at 30°C for 2 h. The rate of fatty acid synthesis was highest at pH 8.5 In presence of 0.4 M Bistris-propane buffer and linear for upto 4 h at 30°C with 80–110 μg plastid protein. Sorbitol was an essential requirement as it prevented the rupturing of leucoplasts by osmosis. ATP and divalent cations were almost absolute requirements, whereas nucleotides, CoA and bicarbonate improved the rate of fatty acid synthesis by two to ten folds. Mg²⁺ and NADH were the preferred cation and nucleotide, respectively. High concentration of dithiothreltol inhibited the incorporation of (¹⁴C) acetate Into fatty acids. The system developed as above could be used for in vitro studies.     

Rheological Properties of Aqueous Dispersions of Xanthan Gum Containing Different Chloride Salts Are Impacted by both Sizes and Net Electric Charges of the Cations

Xanthan gum (XG) is one of the most effective thickener agents used worldwide. In foods products, one of the factors affecting its physical properties is the ionic strength of the medium. Though it is well known that XG rheological properties in aqueous media depend on both type and concentration of electrolytes, correlations between such dispersion properties and molecular aspects of dispersed XG chains are still to be more deeply studied. Thus, in the present study, aqueous XG dispersions [200 mg?(100 mL)^?1] added of Na, K, Mg or Ca chlorides (ionic strength 50 mM or 100 mM) were rheologically characterized, and the corresponding results were explained based on different physicochemical analyses. Comparing to the control (unsalted XG dispersion), KCl and CaCl₂ tended to produce a more drastic decrease of apparent viscosities of XG dispersions than NaCl and MgCl₂. In dynamic-oscillatory assays, the predominance of elastic character over viscous character was more evident for XG dispersions containing KCl and CaCl₂, in particular at frequencies > 0.1 Hz. XG dispersions containing KCl or CaCl₂ also presented smaller pH and |ζ-potentials| values, as well as greater densities and average hydrodynamic diameters of dispersed XG chains, when compared to respective counterparts containing NaCl or MgCl₂. As the decreasing order of the cations radii is K⁺?>?Ca²⁺ ≈ Na⁺?>?Mg²⁺, our results allowed deducing that not only the net electric charges of the cations, but also their sizes, should be considered when analyzing the effect of chloride salts on rheological properties of XG aqueous dispersions, according to the desired for this hydrocolloid (weak thickener, strong thickener or pro-gelling agent).     

Phosphoprotein phosphatase activity of Novikoff hepatoma nucleoli.

Nucleoli from Novikoff hepatoma ascites cells contain phosphatase activity that acts upon ³²P-labeled nucleolar protein substrates. The activity is optimal near pH 7.0 and is inhibited by increasing concentrations of NaCl. The divalent cations CaCl₂, MnCl₂ and CoCl₂ at 6 mM inhibited phosphatase activity from 30–60%. ZnCl₂ completely inhibited the activity above 2 mM while EDTA and MgCl₂ had little effect. The activity was stimulated by dithiothreitol and inhibited by N-ethylmaleimide indicating a requirement for free sulfhydryl groups.     

Identification of brain ecto-apyrase as a phosphoprotein

The divalent cation requirements of NOS activity in bovine retina homogenate supernatant were investigated. Supernatants were assayed under standard conditions (in mM: EDTA 0.45, Ca²⁺ 0.25, Mg²⁺ 4.0). In order to investigate the enzyme's dependence on divalent cations, the tissue homogenate was depleted of di- and trivalent cations by passing it over a cation-exchange column (Chelex 100). Surprisingly, NOS activity was 50-100% higher in this preparation. However, addition of either EDTA (33 M) or EGTA (1 mM) almost fully inhibited NOS activity, suggesting a requirement for residual divalent metal cation(s). Phenanthroline or iminodiacetic acid at low concentrations had little effect on activity, suggesting no requirement for Fe²⁺, Zn²⁺ or Cu²⁺. Ca²⁺ had a moderate stimulatory effect, with an optimum activity around 0.01 mM. Mg²⁺ or Mn²⁺ had little effect at concentrations < 0.25 mM. However, in the presence of EDTA, Mn²⁺ or Ca²⁺ markedly stimulated NOS activity with the optimum at 0.1 mM. At high concentrations (> 0.1-0.2 mM), all divalent cations tested (Ba²⁺, Zn²⁺, Co²⁺, Mn²⁺, Mg²⁺, Ca²⁺), as well as La³⁺, dose-dependently inhibited NOS activity. We propose that retinal NOS requires low concentrations of naturally occurring divalent metal ions, most probably Ca²⁺, for optimal activity and is inhibited by high di- and trivalent metal concentrations, probably by competition with Ca²⁺.     

The fine structure of euchromatin and centromeric heterochromatin in Tenebrio molitor chromosomes

The fine structure of constitutive heterochromatin and euchromatin was compared in electron microscope whole-mount preparations of Tenebrio molitor (Insecta, Coleoptera) spermatocyte nuclei. Tenebrio molitor pachytene chromosomes display extended segments of centromeric heterochromatin and thus are especially suitable for this purpose. When nuclei were incubated in solutions containing different concentrations of NaCl or of MgCl₂, two levels of chromatin fine structures were observed in the euchromatic segments: nucleosome fibers (0.1 mM–20 mM NaCl) and supranucleosomal fibers with 28 nm in diameter (40 mM–100 mM NaCl, 0.2 mM–1.0 mM MgCl₂). The fine structure in the heterochromatic segments was the same as that in the euchromatic segments in all NaCl concentrations and in MgCl₂ concentrations up to 0.4 mM. In higher MgCl₂ concentrations the heterochromatin remained more compact than the euchromatin and consisted of 37-nm-thick fibers in 0.6 mM MgCl₂ and of 65-nm-thick fibers in 1.0 mM MgCl₂. After the 37-nm and the 65-nm fibers had been dispersed in Mg²⁺-free solutions they could be recondensed by incubation in 0.6 mM and 1.0 mM MgCl₂, respectively. It is concluded that a Mg²⁺-sensitive component of the heterochromatin is responsible for the folding of the nucleosome chain to heterochromatin-specific supranucleosomal structures.     

Strontium ranelate stimulates the activity of bone-specific alkaline phosphatase: interaction with Zn2+ and Mg2+

Strontium ranelate (SR) is an orally administered and bone-targeting anti-osteoporotic agent that increases osteoblast-mediated bone formation while decreasing osteoclastic bone resorption, and thus reduces the risk of vertebral and femoral bone fractures in postmenopausal women with osteoporosis. Osteoblastic alkaline phosphatase (ALP) is a key enzyme involved in the process of bone formation and osteoid mineralization. In this study we investigated the direct effect of strontium (SR and SrCl₂) on the activity of ALP obtained from UMR106 osteosarcoma cells, as well as its possible interactions with the divalent cations Zn²⁺ and Mg²⁺. In the presence of Mg²⁺, both SR and SrCl₂ (0.05–0.5 mM) significantly increased ALP activity (15–66 % above basal), and this was dose-dependent in the case of SR. The stimulatory effect of strontium disappeared in the absence of Mg²⁺. The cofactor Zn²⁺ also increased ALP activity (an effect that reached a plateau at 2 mM), and co-incubation of 2 mM Zn²⁺ with 0.05–0.5 mM SR showed an additive effect on ALP activity stimulation. SR induced a dose-dependent decrease in the Km of ALP (and thus an increase in affinity for its substrate) with a maximal effect at 0.1 mM. Co-incubation with 2 mM Zn²⁺ further decreased Km in all cases. These direct effects of SR on osteoblastic ALP activity could be indicating an alternative mechanism by which this compound may regulate bone matrix mineralization.     

Divalent cation-induced interaction of phospholipid vesicle and monolayer membranes

The effects of phospholipid vesicles and divalent cations in the subphase solution on the surface tension of phospholipid monolayer membranes were studied in order to elucidate the nature of the divalent cation-induced vesicle-membrane interaction. The monolayers were formed at the air/water interface. Various concentrations of unilamellar phospholipid (phosphatidylserine, phosphatidylcholine and their mixtures) vesicles and divalent cations (Mg²⁺, Ca²⁺, Mn²⁺, etc.) were introduced into the subphase solution of the monolayers. The changes of surface tension of monolayers were measured by the Wilhelmy plate (Teflon) method with respect to divalent ion concentrations and time.When a monolayer of phosphatidylserine and vesicles of phosphatidylserine/phosphatidylcholine (1 : 1) were used, there were critical concentrations of divalent cations to produce a large reduction in surface tension of the monolayer. These concentrations were 16 mM for Mg²⁺, 7 mM for Sr²⁺, 6 mM for Ca²⁺, 3.5 mM for Ba²⁺ and 1.8 mM for Mn²⁺. On the other hand, for a phosphatidylcholine monolayer and phosphatidylcholine vesicles, there was no change in surface tension of the monolayer up to 25 mM of any divalent ion used. When a phosphatidylserine monolayer and phosphatidylcholine vesicles were used, the order of divalent ions to effect the large reduction of surface tension was Mn²⁺ > Ca²⁺ > Mg²⁺ and their critical concentrations were in between the former two cases. The threshold concentrations also depended upon vesicle concentrations as well as the area/molecule of monolayers. For phosphatidylserine monolayers and phosphatidylserine/phosphatidylcholine (1 : 1) vesicles, above the critical concentrations of Mn²⁺ and Ca²⁺, the surface tension decreased to a value close to the equilibrium pressure of the monolayers within 0.5 h.This decrease in surface tension of the monolayers is interpreted partly as the consequence of fusion of the vesicles with the monolayer membranes. The     

Removal of Cd2+, Pb2+, and Zn2+ from contaminated water using dolomite powder

Dolomite collected from Surat Thani Province in Thailand was investigated for use as a sorbent for the removal of divalent heavy metal cations from an aqueous solution. The sorbent had a surface area of 2.46 m²/g and a pH of zero point charge (pHzpc) of 9.2. Batch sorption was used to examine the effect of the pH (pH 3–7) on the sorption capacity of Cd²⁺, Pb²⁺ and Zn²⁺, alone or together as an equimolar mixture at various concentrations. Alone, each heavy metal cation was adsorbed faster at a higher pH, where the sorption of Cd²⁺ and Pb²⁺ fitted a Langmuir isotherm, but Zn²⁺ sorption best fitted a Freundlich isotherm. Under equimolar competitive sorption, the sorption capacity of each cation was decreased by 75.8% (0.29–0.07 mM/g), 82.8% (0.53–0.09 mM/g), and 95.7% (0.84–0.04 mM/g) for Cd²⁺, Pb²⁺ and Zn²⁺, respectively, compared to that with the respective single cation. Desorption of these heavy metal cations from dolomite was low, with an average desorption level of 0.06–17.4%. Furthermore, since dolomite is readily available and rather cheap, it is potentially suitable for use as an efficient sorbent to sorb Cd²⁺ and Pb²⁺, and perhaps Zn²⁺, from contaminated water.     

Extra-matrix Mg2+ limits Ca2+ uptake and modulates Ca2+ uptake–independent respiration and redox state in cardiac isolated mitochondria

Cardiac mitochondrial matrix (m) free Ca²⁺ ([Ca²⁺]_m) increases primarily by Ca²⁺ uptake through the Ca²⁺ uniporter (CU). Ca²⁺ uptake via the CU is attenuated by extra-matrix (e) Mg²⁺ ([Mg²⁺]_e). How [Ca²⁺]_m is dynamically modulated by interacting physiological levels of [Ca²⁺]_e and [Mg²⁺]_e and how this interaction alters bioenergetics are not well understood. We postulated that as [Mg²⁺]_e modulates Ca²⁺ uptake via the CU, it also alters bioenergetics in a matrix Ca²⁺–induced and matrix Ca²⁺–independent manner. To test this, we measured changes in [Ca²⁺]_e, [Ca²⁺]_m, [Mg²⁺]_e and [Mg²⁺]_m spectrofluorometrically in guinea pig cardiac mitochondria in response to added CaCl₂ (0–0.6 mM; 1 mM EGTA buffer) with/without added MgCl₂ (0–2 mM). In parallel, we assessed effects of added CaCl₂ and MgCl₂ on NADH, membrane potential (ΔΨ_m), and respiration. We found that >0.125 mM MgCl₂ significantly attenuated CU-mediated Ca²⁺ uptake and [Ca²⁺]_m. Incremental [Mg²⁺]_e did not reduce initial Ca²⁺uptake but attenuated the subsequent slower Ca²⁺ uptake, so that [Ca²⁺]_m remained unaltered over time. Adding CaCl₂ without MgCl₂ to attain a [Ca²⁺]_m from 46 to 221 nM enhanced state 3 NADH oxidation and increased respiration by 15 %; up to 868 nM [Ca²⁺]_m did not additionally enhance NADH oxidation or respiration. Adding MgCl₂ did not increase [Mg²⁺]_m but it altered bioenergetics by its direct effect to decrease Ca²⁺ uptake. However, at a given [Ca²⁺]_m, state 3 respiration was incrementally attenuated, and state 4 respiration enhanced, by higher [Mg²⁺]_e. Thus, [Mg²⁺]_e without a change in [Mg²⁺]_m can modulate bioenergetics independently of CU-mediated Ca²⁺ transport.     

Unique Characteristics of Recombinant Hybrid Manganese Superoxide Dismutase from <Emphasis Type="Italic">Staphylococcus equorum and S. saprophyticus</Emphasis>

A recombinant hybrid of manganese dependent-superoxide dismutase of Staphylococcus equorum and S. saprophyticus has successfully been overexpressed in Escherichia coli BL21(DE3), purified, and characterized. The recombinant enzyme suffered from degradation and aggregation upon storage at ?20 °C, but not at room temperature nor in cold. Chromatographic analysis in a size exclusion column suggested the occurrence of dimeric form, which has been reported to contribute in maintaining the stability of the enzyme. Effect of monovalent (Na⁺, K⁺), divalent (Ca²⁺, Mg²⁺), multivalent (Mn^2+/4+, Zn^2+/4+) cations and anions (Cl^?, SO₄ ^2?) to the enzyme stability or dimeric state depended on type of cation or anion, its concentration, and pH. However, tremendous effect was observed with 50 mM ZnSO₄, in which thermostability of both the dimer and monomer was increased. Similar situation was not observed with MnSO₄, and its presence was detrimental at 200 mM. Finally, chelating agent appeared to destabilize the dimer around neutral pH and dissociate it at basic pH. The monomer remained stable upon addition of ethylene diamine tetraacetic acid. Here we reported unique characteristics and stability of manganese dependent-superoxide dismutase from S. equorum/saprophyticus.     

Aggregation of mono- and dinucleosomes into chromatin-like fibers

Three classes of chicken erythrocyte chromatin particles differing in their content of lysine-rich histones and/or spacer DNA have been studied in order to determine their ability to aggregate into complexes resembling those observed in native chromatin. The complexes have been obtained in the presence of MgCl₂ and NaCl and studied by electron microscopy. Mononucleosomes, containing spacer DNA and histones H1 and H5, give rise to thick (about 70 nm) ellipsoidal particles in the presence of 0.5 mM MgCl₂. These particles are disrupted by the addition of small amounts of NaCl (5–20 mM). On the other hand in 0.5 mM MgCl₂ dinucleosomes give rise to regular fibrous complexes of about 40 nm in diameter which are very similar to native chromatin fibers. These complexes are much more stable when NaCl is added. We conclude that for the stability of nucleosomal aggregates, similar to native chromatin fibers, a continuity of DNA structure is not required, but the presence of divalent cations, spacer DNA and lysine-rich histones is essential.     

Binding citrate/DNA in presence of divalent cations – Potential mimicry of acidic peptides/DNA interactions

Citric acid whose structure is comparable to that of small acidic peptides, can bind to DNA in the presence of divalent cations (Cu²⁺, Fe²⁺, Zn²⁺, Mg²⁺). Citrate-DNA interaction occurs also in a cell homogenate and in this experimental model too requires the presence of natural divalent cations. In fact the addition of 2 mM EDTA to cell homogenate strongly decreases the DNA-citrate binding. The results demonstrate that divalent cations can act as bridges between two acidic molecules and that citric acid can mimic the structure of acidic peptides.