Protection of DNA by salts against thermodegradation at temperatures typical for hyperthermophiles

The effect of physiological concentrations of KCl and MgCl₂ on the chemical stability of double-stranded and single-stranded DNA has been studied at temperatures typical for hyperthermophiles. These two salts protect both double and single-stranded DNA against heat-induced cleavage by inhibiting depurination. High KCl concentrations also protect DNA cleavage at apurinic sites, while high MgCl₂ concentrations stimulate this cleavage. It has been previously proposed that salt protects double-stranded DNA against depurination by stabilizing the double helix. However, the inhibition of the depurination of single-stranded DNA by KCl and MgCl₂ indicates that this effect is more probably due to a direct interaction of salts with purine nucleotides. These results suggest that the number and nature of heat-induced DNA lesions which have to be repaired might be quite different from one hyperthermophile to another, depending on their intracellular salt concentration. High salt concentrations might be also useful to protect DNA in long polymerase chain reaction (PCR) experiments and for long-term preservation. Received: October 12, 1997 / Accepted: January 29, 1998     

MICRURGICAL STUDIES IN CELL PHYSIOLOGY : V. THE ANTAGONISM OF CATIONS IN THEIR ACTIONS ON THE PROTOPLASM OF AM?BA DUBIA.

I. The Plasmalemma. 1. On the plasmalemma of amebæ CaCl₂ antagonizes the toxic action of LiCl better than it does NaCl, and still better than it does KCl. MgCl₂ antagonizes the toxic action of NaCl better than it does LiCl and still better than it does KCl. 2. CaCl₂ antagonizes the toxic action of LiCl and of KCl better than does MgCl₂: MgCl₂ antagonizes NaCl better than does CaCl₂. II. The Internal Protoplasm. 3. The antagonizing efficiency of CaCl₂ and of MgCl₂ are highest against the toxic action of KCl on the internal protoplasm, less against that of NaCl, and least against that of LiCl. 4. CaCl₂ antagonizes the toxic action of LiCl better than does MgCl₂: MgCl₂ antagonizes the toxic action of NaCl and of KCl better than does CaCl₂. 5. LiCl antagonizes the toxic action of MgCl₂ on the internal protoplasm more effectively than do NaCl or KCl, which have an equal antagonizing effect on the MgCl₂ action. III. The Nature of Antagonism. 6. When the concentration of an antagonizing salt is increased to a toxic value, it acts synergistically with a toxic salt. 7. No case was found in which a potentially antagonistic salt abolishes the toxic action of a salt unless it is present at the site (surface or interior) of toxic action. 8. Antagonistic actions of the salts used in these experiments are of differing effectiveness on the internal protoplasm and on the surface membrane.     

THE PROTEIN COMPOSITION OF ISOLATED MYELIN1

—Three fractions, each containing markedly different proteins, was obtained from myelin: (1) The first fraction was obtained as an insoluble residue when myelin was extracted with neutral chloroform-methanol (CM, 2:1, v/v). It was digestible with trypsin and had an amino acid composition similar to that of the acidic proteolipid protein of Wolfgkam (1966). (2) The second fraction was obtained as a precipitate by the addition of various electrolytes (KCl, NaCl, CaCl₂, MgCl₂ or HCl) to the CM (2:1 v/v) extract. This fraction consisted mainly of a basic protein which exhibited an electrophoretic mobility and amino acid composition indistinguishable from those of the basic protein obtained from white matter (Martensson and LeBaron, 1966). This procedure provided for a simple and rapid isolation of the basic protein from myelin. Depending on the conditions of precipitation, this fraction was either free of lipid or contained tri- and diphosphoinositide. The effects of different ions at differing concentrations and the yield and nature of the precipitate have been studied. (3) A third fraction remained in solution in CM (2:1, v/v) after the addition of the electrolyte. It comprised the bulk of the myelin lipids and a protein fraction which was resistant to digestion with trypsin and had an amino acid composition similar to the classical proteolipid protein of Folch-Pi and Lees (1951). The possibility of a salt-type bonding between the basic protein and the polyphosphoinositides is discussed, and values for tri- and diphosphoinositide in bovine myelin are given.     

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).     

THE EFFECT OF CERTAIN ELECTROLYTES AND NON-ELECTROLYTES ON PERMEABILITY OF LIVING CELLS TO WATER

1. Permeability to water in unfertilized eggs of the sea urchin, Arbacia punctulata, is found to be greater in hypotonic solutions of dextrose, saccharose and glycocoll than in sea water of the same osmotic pressure. 2. The addition to dextrose solution of small amounts of CaCl₂ or MgCl₂ restores the permeability approximately to the value obtained in sea water. 3. This effect of CaCl₂ and MgCl₂ is antagonized by the further addition of NaCl or KCl. 4. It is concluded that the NaCl and KCl tend to increase the permeability of the cell to water, CaCl₂ and MgCl₂ to decrease it. 5. The method here employed can be used for quantitative study of salt antagonism.     

A thermally induced alteration in lysosome membranes: Salt permeability at 0 and 37 °C

Preparations of radioactive lysosomes were obtained from mouse kidney after injection of radioactive iodine-labeled bovine ribonuclease. Stability of these lysosomes in various media was estimated from measurements of proteolytic activity towards the ribonuclease, and of ribonuclease retention in particles. The lysosomes were stable at 37 °C in isotonic, sucrose-free solutions of KCl, NaCl and potassium acetate, and in mixtures of these with MgCl₂, showing that these salts are relatively impermeant through the lysosomal membranes. The membranes were less permeable to Na⁺ than to K⁺. Both KCl and NaCl exerted their optimal protective effects over a broad concentration range above 0.125 M in 0.025 M acetate buffer. Mg²⁺ enhanced the protective effect of both K⁺ and Na⁺; the osmotic effect of 0.075 M NaCl-0.05 M MgCl₂ was indistinguishable during the entire course of ribonuclease digestion from that of isotonic sucrose. Osmotic protection by KCl-MgCl₂ was demonstrated over the pH range 5.5–7.0. A marked alteration in membrane properties occurs at lower temperatures in 0.11 M KCl-0.01 M MgCl₂ such that, at 0 °C, K⁺ permeability is much higher than at 37 °C, as shown by a several-fold decrease in stability at the lower temperature.     

Effect of metal ions on glucose-6-phosphate dehydrogenase of fungi,bacteria, and rat

We have determined the effects of KCl, ZnCl₂, and MgCl₂ on kinetic parameters of glucose-6-phosphate dehydrogenases from the bacteriaEscherichia coli, Bacillus stearothermophilus, andLeuconostoc mesenteroides, from the fungiAspergillus parasiticus, Alternaria alternata, Aphanomyces astaci, Saccharomyces cerevisiae, andTorula sp., and from the mammalRattus rattus. Each enzyme was stimulated by increasing ionic strength (KCl) and by MgCl₂. One bacterial enzyme, fromE. coli, three fungal enzymes, fromA. parasiticus, S. cerevisiae, andTorula sp., and the rat liver enzyme were inhibited by ZnCl₂. These data are discussed in light of our previous proposal that Zn²⁺ inhibition of this enzyme may stimulate versicolorin synthesis byA. parasiticus.     

Influence of the explantation milieu on intranuclear (Na), (K) and (Mg) of Chironomus thummi salivary gland cells

Intranuclear Na, K and Mg concentrations were determined in cells of salivary glands incubated for 1^h in selected NaCl/KCl/MgCl₂ media. By variation of the external milieu beyond “physiological” limits the intranuclear electrolytes can be shifted between ca 100 and 280 mM [K]ⁱ, between ca 8 and 100 mM [Na]ⁱ and between ca 5 and 75 mM [Mg]ⁱ. No significant competition or interactions of the 3 ionic species are apparent. The relationships [K]^e : [K]ⁱ and [Na]^e : [Na]ⁱ can best be described by a positive and linear, that between [Mg]^e : [Mg]ⁱ by a negative and exponential function. Regression parameters are given which permit a computation of intranuclear [Na], [K] and [Mg] as induced by NaCl/KCl/MgCl₂ in any binary or triple combination that is tolerated by the explanted gland without visible damage.     

Nuclear and polyribosomal ribonucleoprotein particles containing poly(A) in rat liver cells

Poly(A) containing ribonucleoprotein particles were prepared from rat liver nuclei and polyribosomes. The particles have sedimentation coefficients of 14 S and 9 S, respectively. In Cs₂SO₄ density gradients the particles banded at densities of 1.28–1.29 g cm^-3. Both nuclear and polyribosomal poly(A)-RNP contain in addition to some minor polypeptides, two main polypeptides having molecular weights of 63 000 and 90 000 dalton, respectively indistinguishable from each other according to their electrophoretic mobilities.Abbreviations STKM 0.25 M sucrose, 0.05 M Tris-HCl, pH 7.2, 0.025 M KCl, 0.005 M MgCl₂ - TKM 0.05 M Tris-HCl, pH 7.5, 0.025 M KCl, 0.005 M MgCl₂ - STM II 0.1 M NaCl, 0.01 M Tris-HCl, pH 8, 0.001 M MgCl₂ - DTT dithiothreitol - SDS sodium dodecylsulphate     

Ethylene and ethane production in response to salinity stress

Abstract Ethylene and ethane production in mung bean hypocotyl sections were evaluated as possible indicators of stress due to contact with four salts that are common in natural sites. Ethylene production decreased with increasing concentrations of applied NaCl and KCl. When CaCl₂ was applied, the ethylene evolution was greater. However, when MgCl₂ was applied, ethylene evolution remained high then decreased and at higher salt concentrations again showed an increase. NaCl (up to 0.1 kmol m^?1) and KCl (up to 0.5 kmol m^?3) caused a concentration-dependent increase in ethane production. The ethane production with CaCl₂ was the lowest among the salts tested and only a minute increase was noticed with the increase of concentration from 0.01 to 1 kmol m^?3. Ethane production showed a distinct maximum at 0.2 kmol m^?3 MgCl₂. The introduction of 0.01 kmol m^?3 CaCl₂, as well as anaerobic conditions obtained by purging vials with N₂, eliminated that high ethane production. Respiratory activity of the mung bean hypocotyl sections in MgCl₂ concentrations from 0 to 0.5 kmol m^?3 was correlated with ethane but not with ethylene production. The ethane/ethylene ratio showed three patterns for the four salts tested.     

Dissociation of N(2) Gas-induced Monomeric Ribosomes and Functioning of the Derived Subunits in Protein Synthesis in Pea

The dissociation of N₂ gas-induced monomeric ribosomes from the pea root was studied by varying the concentration of KCl (or NH₄Cl) and MgCl₂ in the presence of dithiothreitol. These monoribosomes were shown to dissociate completely into subunits at 0.5m KCl or NH₄Cl in the presence of 5 mm MgCl₂. The 40S subunits were more susceptible to structural change in KCl than were the 60S subunits. On the other hand, the 60S subunits appeared to be more labile to NH₄Cl.     

Studies on the effects of certain salts on germination,on growth of root,and on metabolism

Summary The effects of different concentrations of KCl, K₂SO₄, MgCl₂ and MgSO₄ on the growth in length of the first seminal root of wheat, and on the change in fresh and oven-dry weight of the seedling and its component parts have been studied. The effect of mannitol was also investigated for comparison and to study the osmotic action. The effect of salts on root growth was dependent on salt species; all effects were specific to ions and not due to osmotic activity of solution. The growth of wheat roots was suppressed by concentrations of salts much lower than those required to suppress germination. All solutions of KCl from 0.1 to 50 me/l checked the growth of the root; the retardation increased with increase of concentration. In K₂SO₄ there was a slight activation of root growth for one day in 0.1 and 0.5 me/l; then the growth was suppressed after that. In all other concentrations from 1 to 50 me/l the growth was retarded. In MgCl₂ or MgSO₄ there was some activation of root elongation in 0.05, 0.1 and 0.5 me/l; but higher concentrations retarded root growth.     

Two mechanisms for spreading depression in the chicken retina

Two mechanisms have been proposed to explain spreading depression (SD): one based on a release of glutamate (Van Harreveld, 1959), and the other on a release of potassium (Grafstein, 1956) from neuronal elements. Both glutamate and KCl cause transparency changes in the retina, comparable to those occurring in this tissue during SD. The glutamate effect is inhibited by MgCl₂ (10 mM), in contrast to the transparency change due to KCl which is not affected by Mg⁺⁺. Also SD is usually inhibited by MgCl₂ which suggests that such SDs are based on a glutamate release. Impairment of the tissue metabolism promotes SDs which are insensitive to MgCl₂. The resulting failure of the mechanisms that transport K⁺ and glutamate which leak out of the intracellular compartment back into the cells and fibers, seems to be involved in the generation of Mg⁺⁺ insensitive SDs. This may facilitate either K-based SDs or glutamate-based SDs since the inhibitory effect of Mg⁺⁺ is counteracted by an enhanced glutamate concentration. Both proposed mechanisms for SD seem to be possible under special circumstances.     

Reversible abolition of enhancement in isolated spinach chloroplasts

The photosynthetic evolution of oxygen by isolated chloroplasts of Spinacia oleracea L. was studied using a modulated oxygen electrode. The enhancement effect, measured as the increase in the relative quantum yield of 650-nanometer light due to the presence of 710-nanometer light, was profoundly influenced by the concentration of inorganic cations in the bathing medium. Chloroplast fragments immersed in a solution containing a very low concentration of MgCl₂ or KCl, did not display enhancement but could be made to do so by raising the concentration of MgCl₂ to 3 mm, or that of KCl to 35 mm. This change in the enhancement properties was completely reversible. The maximum value of enhancement in a MgCl₂ solution appeared to occur at a concentration between 15 and 30 mm, while in KCl, the enhancement effect increased almost linearly up to a concentration of 100 mm.     

ELECTROKINETICS : XVII. SURFACE CHARGE AND ION ANTAGONISM

1. The question of the critical pore diameter for streaming potential is discussed. 2. The surface charge is calculated for cellulose in contact with solutions of K₃PO₄, K₂CO₃, K₂SO₄, KCl, and ThCl₄. 3. The surface charge of cellulose in contact with a solution of 2 x 10^–4 N NaCl is calculated as a function of temperature and is found to show a sharp break at 39°. This is interpreted in terms of the change of the specific heat of water. 4. A marked ion antagonism is found in NaCl:KCl, KCl:MgCl₂, NaCl:MgCl₂, NaCl:CaCl₂, KCl:CaCl₂, CaCl₂:MgCl₂ mixtures when the surface charge is calculated as a function of concentration.     

Incorporation of 14C-Tyrosine into Soluble Ribonucleic Acid from Baker’s Yeast

The incorporation of ¹⁴C-tyrosine into S-RNA catalyzed by a partially purified tyrosine activating enzyme from baker’s yeast was observed. The maximum incorporation was shown in the presence of 5 μmoles of ATP, 10 μmoles of MgCl₂ and 10~100 μmoles of KCl in the reaction mixture of total volume of 1ml, at pH 7.8 when 1.2 mg of S-RNA, 0.1 μmole of ¹⁴C-tyrosine and 400 μg of enzyme protein were used. Beyond the concentration of ATP, MgCl₂ and KCl described above, the tendency of inhibition was observed. The incorporation was strongly inhibited by pCMB and reactivated by cysteine. Manganese and calcium ions were effective as substitutes for magnesium. S-RNA used was prepared from whole baker’s yeast cell with phenol, but S-RNA obtained from the supernatant of the ground yeast had lost its incorporating activity.     

Loose association of ribulose 1,5-bisphosphate carboxylase/oxygenase with chloroplast thylakoid membranes

The intra-chloroplastic distribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) between thylakoid membranes and stroma was studied by determining the enzyme activities in the two fractions, obtained by the rapid centrifugation of hypotonically disrupted chloroplast preparations of spinach and pea leaf tissues. The membrane-associated form of RuBisCO was found to increase in proportion to the concentration of MgCl₂ in the disrupting medium; with 20 mM MgCl₂ approximately 20% of the total RuBisCO of spinach chloroplasts and 10% of that of pea chloroplasts became associated with thylakoid membranes. Once released from membranes in the absence of MgCl₂, addition of MgCl₂ did not cause reassociation of the enzyme. The inclusion of KCl in the hypotonic disruption buffer also caused the association of RuBisCO with membranes; however, up to 30 mM KCl, only minimal enzyme activities could be detected in the membranes, whereas above 40 mM KCl there was a sharp increase in the membrane-associated form of the enzyme.Higher concentrations of chloroplasts during the hypotonic disruption, as well as addition of purified preparations of RuBisCO to the hypotonic buffer, resulted in an increase of membrane-associated activity. Therefore, the association of the enzyme with thylakoid membranes appears to be dependent on the concentration of RuBisCO. P-glycerate kinase and aldolase also associated to the thylakoid membranes but NADP-linked glyceraldehyde-3-P dehydrogenase did not. The optimal conditions for enzyme association with the thylakoid membranes were examined; maximal association occurred at pH 8.0. The association was temperature-insensitive in the range of 4° to 25° C. RuBisCO associated with the thylakoid membranes could be gradually liberated to the soluble form upon shaking in a Vortex mixer at maximal speed, indicating that the association is loose.Abbreviations DTT dithiothreitol - RuBP ribulose 1,5-bisphosphate - RuBisCO ribulose 1,5-bisphosphate carboxylase/oxygenase - MES 2-(N-morpholino) ethane sulfonic acid     

Effects of calcium and kinetin on growth and cell wall composition of pea epicotyls

Kinetin and CaCl₂, in the presence of indoleacetic acid, promoted lateral expansion of epicotyls of decapitated and derooted Alaska pea seedlings (Pisum sativum L.) and inhibited their elongation. This growth response was correlated with the development of cell walls unusually rich in pectic uronic acids. Epicotyls in calcium-auxin solutions continued to enlarge and to add new wall material long after tissues in auxin only had stopped. Longitudinal enlargement, associated with the development of walls poor in pectic uronic acids, was favored by KCl, MgCl₂, and ethylenediaminetetraacetate. The last of these agents promoted the loss of ⁴⁵Ca from the epicotyls. Seedings grown in vermiculite moistened with CaCl₂, KCl, or MgCl₂ solutions did not differ in appearance or in the composition of their walls. They responded similarly to experimental treatment except that the decapitated epicotyls of the MgCl₂-grown plants suffered an absolute loss of pectic uronate when incubated in that salt.     

Substituting manganese for magnesium alters certain reaction properties of the (Na+ + K+)-ATPase

MnCl₂ was partially effective as a substitute for MgCl₂ in activating the K⁺-dependent phosphatase reaction catalyzed by a purified (Na⁺ + K⁺)-ATPase enzyme preparation from canine kidney medulla, the maximal velocity attainable being one-fourth that with MgCl₂. Estimates of the concentration of free Mn²⁺ available when the reaction was half-maximally stimulated lie in the range of the single high-affinity divalent cation site previously identified (Grisham, C.M. and Mildvan, A.S. (1974) J. Biol. Chem. 249, 3187–3197). MnCl₂ competed with MgCl₂ as activator of the phosphatase reaction, again consistent with action through a single site. However, with MnCl₂ appreciable ouabaininhibitable phosphatase activity occurred in the absence of added KCl, and the apparent affinities for K⁺ as activator of the reaction and for Na⁺ as inhibitor were both decreased. For the (Na⁺ + K⁺)-ATPase reaction substituting MnCl₂ for MgCl₂ was also partially effective, but no stimulation in the absence of added KCl, in either the absence or presence of NaCl, was detectable. Moreover, the apparent affinity for K⁺ was increased by the substitution, although that for Na⁺ was decreased as in the phosphatase reaction. Substituting MnCl₂ also altered the sensitivity to inhibitors. For both reactions the inhibition by ouabain and by vanadate was increased, as was binding of [⁴⁸V]-vanadate to the enzyme; furthermore, binding in the presence of MnCl₂ was, unlike that with MgCl₂, insensitive to KCl and NaCl. Inhibition of the phosphatase reaction by ATP was decreased with 1 mM but not 10 mM KCl. Finally, inhibition of the (Na⁺ + K⁺)-ATPase reaction by Triton X-100 was increased, but that by dimethylsulfoxide decreased after such substitution.