23Na NMR investigation of the interaction between DNA and divalent metallic cations

The aim of this study is to follow the thermodynamic behaviour of Na⁺ ions, acting as natural counterions of DNA, in the presence of divalent metal ions, by using the²³Na NMR technique. With the help of the²³Na entropy of fluctuations concept introduced by Lenk, we propose the following decreasing sequence: Mg⁺⁺, Zn⁺⁺, Cd⁺⁺, Mn⁺⁺, and Cu⁺⁺, for the magnitude of divalent metal ions interactions with DNA phosphate sites.     

Dielectric properties of polyelectrolytes. III. Effect of divalent cations on dielectric increment of polyacids

Dielectric dispersion of polyacrylic acid (PAA) and polystyrene sulfonic acid (PSS) was measured in the presence of divalent cations. Effects of divalent ions were studied by neutralization with varying ratios of sodium hydroxide and divalent base concentration, addition of salts of divalent cations, and neutralization with divalent bases only. Two dispersion regions were observed in all cases, i.e., low-frequency dispersion (10²–10⁴ Hz) and high-frequency dispersion (10⁵–10⁶ Hz). The dielectric increment increases in the presence of sodium and alkaline earth metal ions together, but not with sodium and transition metal ions. This is due to the increment of low-frequency dispersion and is attributable to the fluctuation of bound counterions which is explained by our theory previously reported.¹ In the case of PAA neutralized with large fractions of divalent ions, or with divalent ions only, the increment is very small because of reduction of the fluctuation by interaction between bound ions at the neighboring sites and reduction of the effective length of polyion probably due to chelation by divalent ions. There are some differences among the effects of Mg⁺⁺, Ca⁺⁺, and Ba⁺⁺ on dielectric increment which may result from affinity or chelating ability of these ions.     

Muscle: A Three Phase System : The partition of divalent ions across the membrane

The partition of sulfate, Ca⁺⁺, and Mg⁺⁺ across the membrane of the sartorius muscle has been studied, and the effect of various concentrations of these ions in the Ringer solution on the cellular level of Na⁺, K⁺, and Cl^- has been determined. The level of the three divalent ions in toad plasma and muscle in vivo has been assayed. Muscle was found to contain an almost undetectable amount of inorganic sulfate. Increases in the external level of these ions brought about increases in intracellular content, calculated from the found extracellular space as determined with radioiodinated serum albumin or inulin. Less of the cell water is available to sulfate than to Cl^-, and the Mg⁺⁺ space is less than the Na⁺ space. An amount of muscle water similar to that found for Li⁺ and I^- appears to be available to these divalent ions. Sulfate efflux from the cell was extremely rapid, and it was not found possible to differentiate kinetically between intra- and extracellular material. These results are consistent with the theory of a three phase system, assuming the muscle to consist of an extracellular phase and two intracellular phases. Mg⁺⁺ and Ca⁺⁺ are adsorbed onto the ordered phase, and increments in cellular content found on raising the external level are assumed to occur in the free intracellular phase.     

The effect of divalent metal ions on the activity of Mg++ depleted ribulose-1,5-bisphosphate oxygenase

Ribulose-1,5-bisphosphate carboxylase-oxygenase is deactivated by removal of Mg⁺⁺. The enzyme activities can be restored to a different extent by the addition of various divalent ions in the presence of CO₂. Incubation with Mg⁺⁺ and CO₂ restores both enzyme activities, whereas, the treatment of the enzyme with the transition metal ions (Mn⁺⁺, Co⁺⁺, and Ni⁺⁺) and CO₂ fully reactivates the oxygenase: however, the carboxylase activity remains low. In experiments where CO₂-free conditions were conscientiously maintained, no reactivation of RuBP oxygenase was observed, although Mn⁺⁺ ions were present. Other divalent cations such as Ca⁺⁺ and Zn⁺⁺, restore neither the carboxylase nor the oxygenase reaction. Furthermore, the addition of Mn⁺⁺ to the Mg⁺⁺ and CO₂ preactivated enzyme significantly inhibited carboxylase reactions, but increased the oxygenase reaction.Abbreviation RuBP ribulose-1,5-bisphosphate. The enyme unit for RuBP carboxylase is defined as mol CO₂ fixed·min^-1 and for the RuBP oxygenase as mol O₂ consumed · min^-1     

Further Studies on the Roles of Sodium and Potassium in the Generation of the Electro-Olfactogram : Effects of mono-, di-, and trivalent cations

In the negative EOG-generating process a cation which can substitute for Na⁺ was sought among the monovalent ions, Li⁺, Rb⁺, Cs⁺, NH₄⁺, and TEA⁺, the divalent ions, Mg⁺⁺, Ca⁺⁺, Sr⁺⁺, Ba⁺⁺, Zn⁺⁺, Cd⁺⁺, Mn⁺⁺, Co⁺⁺, and Ni⁺⁺, and the trivalent ions, Al⁺⁺⁺ and Fe⁺⁺⁺. In Ringer solutions in which Na⁺ was replaced by one of these cations the negative EOG's decreased in amplitude and could not maintain the original amplitudes. In K⁺-Ringer solution in which Na⁺ was replaced by K⁺, the negative EOG's reversed their polarity. Recovery of these reversed potentials was examined in modified Ringer solutions in which Na⁺ was replaced by one of the above cations. Complete recovery was found only in the normal Ringer solution. Thus, it was clarified that Na⁺ plays an irreplaceable role in the generation of the negative EOG's. The sieve hypothesis which was valid for the positive EOG-generating membrane or IPSP was not found applicable in any form to the negative EOG-generating membrane. The reversal of the negative EOG's found in K⁺- , Rb⁺- , and Ba⁺⁺-Ringer solutions was attributed to the exit of the internal K⁺. It is, however, not known whether or not Cl^- permeability increases in these Na⁺-free solutions and contributes to the generation of the reversed EOG's.     

tRNA structure and binding sites for cations

Equilibrium dialysis and electronic and nuclear resonance spectroscopy show that tRNA cooperatively binds divalent metal ions at very low concentrations (free metal concentration 3 × 10 ^?6 M). The first two methods show that different purified tRNAs have a very similar behavior, including initiator tRNA_F^met. tRNAs with an extra arm in the clover-leaf model, however, appear to have a slightly different behavior. The binding can be described in terms of two classes of sites. The cooperative association of divalent ions binding first does not parallel a cooperative change in the hyperchromism of the tRNA, while the non-cooperative association of the second class of divalent ions corresponds to the concentrations needed to obtain a cooperative melting of the tRNA. The temperature dependence of the number of binding sites and of their binding constants is also presented. The nature of the divalent ion gives the following efficiency: for the cooperativity Co⁺⁺>Mg⁺⁺>Mn⁺⁺ for the weak binding sites Mn⁺⁺>Co⁺⁺>Mg⁺⁺     

Abnormal lysosomal hydrolases excreted by cultured fibroblasts in I-cell disease (mucolipidosis II).

The characteristics of rat liver mitochondria swelling induced by diamide, an oxidizing agent for thiol groups, and by Ca ions are very similar. In both cases the swelling, which is initiated by addition of 0.5–1 mM phosphate or acetate, is prevented by FCCP, antimycin A, EGTA, Mg⁺⁺ and ruthenium red. Diamide potentiates the swelling action of Ca⁺⁺, while DTE potentiates that of Mg⁺⁺. The additive effects of calcium and diamide on rat liver mitochondria have been correlated with their synergic action in promoting the release of mitochondrial Mg⁺⁺. The results strongly indicate that some of the effects of diamide are mediated by a mobilization of endogenous divalent ions and that the antagonism between Ca⁺⁺ and Mg⁺⁺ is closely correlated with the redox state of membrane bound thiol groups.     

Graded and All-or-None Electrogenesis in Arthropod Muscle : I. The effects of alkali-earth cations on the neuromuscular system of Romalea microptera

Graded electrically excited responsiveness of Romalea muscle fibers is converted to all-or-none activity by Ba⁺⁺, Sr⁺⁺, or Ca⁺⁺, the two former being much the more effective in this action. The change occurs with as little as 7 to 10 per cent of Na⁺ substituted by Ba⁺⁺. The spikes now produced have overshoots and may be extremely prolonged, lasting many seconds. During the spike the membrane resistance is lower than in the resting fiber, but the resting resistance and time constant are considerably increased by the alkali-earth ions. The excitability is also increased, spikes arising neurogenically from spontaneous repetitive discharges in the axon as well as myogenically from spontaneous activity in the muscle fibers. Repetitive responses frequently occur on intracellular stimulation with a brief pulse. The data indicate that the alkali-earth ions exert a complex of effects on the different action components of electrically excitable membrane. They may be described in terms of the ionic theory as follows: The resting K⁺ conductance is diminished. The sodium inactivation process is also diminished, and sodium activation may be increased. Together these changes can act to convert graded responsiveness to the all-or-none variety. The alkali-earth ions can also to some degree carry inward positive charge during activity, since spikes are produced when Na⁺ is fully replaced with the divalent ions.     

CHARACTERIZATION OF LYMPHOCYTE TRANSFORMATION INDUCED BY ZINC IONS

Lymphocyte cultures from all normal human adults are stimulated by zinc ions to increase DNA and RNA synthesis and undergo blast transformation. Optimal stimulation occurs at 0.1 mM Zn⁺⁺. Examination of the effects of other divalent cations reveals that 0.01 mM Hg⁺⁺ also stimulates lymphocyte DNA synthesis. Ca⁺⁺ and Mg⁺⁺ do not affect DNA synthesis in this culture system, while Mn⁺⁺, Co⁺⁺, Cd⁺⁺, Cu⁺⁺, and Ni⁺⁺ at concentrations of 10^-7–10^-3 M are inhibitory. DNA and RNA synthesis and blast transformation begin to increase after cultures are incubated for 2–3 days with Zn⁺⁺ and these processes reach a maximum rate after 6 days. The increase in Zn⁺⁺-stimulated lymphocyte DNA synthesis is prevented by rendering cells incapable of DNA-dependent RNA synthesis with actinomycin D or by blocking protein synthesis with cycloheximide or puromycin. Zn⁺⁺-stimulated DNA synthesis is also partially inhibited by 5'-AMP and chloramphenicol. Zn⁺⁺ must be present for the entire 6-day culture period to produce maximum stimulation of DNA synthesis. In contrast to its ability to independently stimulate DNA synthesis, 0.1 mM Zn⁺⁺ inhibits DNA synthesis in phytohemagglutinin-stimulated lymphocytes and L1210 lymphoblasts.     

Selection and characterization of cadmium-resistant suspension cultures of the wild tomato Lycopersicon peruvianum

Suspension cultures of Lycopersicon peruvianum were selected for resistance to cadmium by stepwise exposure to increasing concentrations of cadmium sulfate. Resistant cells grow in 1500 micromolar Cd⁺⁺. This resistance was retained for thirty generations without selection. Both resistant and parental sensitive cultures take up Cd⁺⁺ at similar rates and to the same final levels. Exposure of sensitive or resistant cultures to Cd⁺⁺, Cu⁺⁺, or Zn⁺⁺ leads to the intracellular accumulation of a low molecular weight, cysteine-rich, cadmium-binding protein. This metallothionein is induced over fifteen fold by 100 M cadmium and builds up to about five fold higher levels in the resistant cultures.Abbreviations Cd⁺⁺ divalent cadmium ion - Cu⁺⁺ divalent copper ion - Zn⁺⁺ divalent zinc ion - BA benzyl adenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-acetic acid     

The penetration of some cations into muscle

The influx of Na⁺, K⁺, Rb⁺, and Cs⁺ into frog sartorius muscle has been followed. The results show that a maximum rate is found for K⁺, while Na⁺ and Cs⁺ penetrate much more slowly. Similar measurements with Ca⁺⁺, Ba⁺⁺, and Ra⁺⁺ show that Ba⁺⁺ penetrates at a rate somewhat greater than that of either Ca⁺⁺ or Ra⁺⁺. All these divalent cations, however, penetrate at rates much slower than do the alkali cations. The results obtained are discussed with reference to a model that has been developed to explain the different penetration rates for the alkali cations.     

Studies on Peptidases of Aspergillus oryzae

The homogeneity of Aspergillus dipeptidase prepared according to the standard method established by us was ascertained by ultracentrifugation and some characteristic properties of the enzyme was further investigated.

Hydrolysis of various dipeptides by the purified dipetidase was tested in the presence of divalent metal ions such as Co⁺⁺ or Zn⁺⁺, and the characteristics of greatest interest may be enumerated as follows:

1. The homogeneous dipeptidase requires Zn⁺⁺ for activation in the case of the hydrolysis of leucylglycine, leucylalanine leucylleucine, etc.

2. The homogeneous dipeptidase requires Co⁺⁺ for activation in the case of the hydrolysis of glycylleucine, glycylleucine, glycylglycine, glycylphenylalanine, etc.

3. In the case of the hydrolysis of alanylglycine, alanylleucine, valylglycine, etc., this enzyme does not require any metal ions.

     

Cation dependence of guinea pig epidermal cell spreading

Summary To understand the earliest phases of epidermal cell spreading we have sought a defined in vitro system. We studied the divalent cation dependence of guinea pig epidermal cell spreading in media containing varying concentrations of cations. No spreading occurred in calcium-magnesium-free Dulbecco's modified Eagle's medium (CMF-DME) in the presence of cation-free fetal bovine serum; however, significant spreading occurred if the medium was supplemented with Mg⁺⁺ plus Ca⁺⁺ or Mg⁺⁺ alone. Supplementing with Ca⁺⁺ alone led to much less spreading. These cations in CMF-DME did not support spreading in the absence of serum or the presence of serum albumin. Assaying cell spreading in a simple salt solution consisting of NaCl, KCl, Tris buffer, pH 7.4 plus dialyzed serum and a series of divalent cation supplements (Ca⁺⁺, Mg⁺⁺, Mn⁺⁺, Co⁺⁺, Zn⁺⁺, Ni⁺⁺), showed that only Mg⁺⁺ and Mn⁺⁺, and to a lesser extent, Ca⁺⁺, supported cell spreading. In contrast to Mg⁺⁺, however, Mn⁺⁺ could support spreading in the absence of whole serum if serum albumin were present. Although Mn⁺⁺ plus serum albumin supported more rapid spreading at lower cation concentrations than Mg⁺⁺ plus serum, equal concentrations of Ca⁺⁺ completely blocked the Mn⁺⁺ effect. In contrast to the increasing cell spreading, which occurred in Mg⁺⁺-containing medium with time, cell death occurred in Mn⁺⁺-containing medium by 24 h. Consonant with studies from other laboratories, human foreskin fibroblasts spread in Mn⁺⁺-containing salt solution in the absence of protein supplements. These experiments indicate for epidermal cell spreading that Mg⁺⁺ is the important cation in tissue culture media, that under proper cation conditions epidermal cells do not need a specific spreading protein (i.e. a protein that has been demonstrated to support cell spreading), that Mn⁺⁺ and Mg⁺⁺-induced spreading seem to represent different mechanisms, that fibroblastic and epidermal cells have different cation requirements for in vitro spreading, and that the crucial role cations play in cell spreading remains to be elucidated. This work was supported in part by Public Health Service grant CA34470-01 (KSS) awarded by the National Cancer Institute, Bethesda, Md.     

QUANTITATIVE STUDIES OF PHAGOCYTOSIS : Kinetic Effects of Cations and Heat-Labile Opsonin

Kinetic analysis of the initial ingestion rate of albumin-coated paraffin oil particles by human granulocytes and rabbit alveolar macrophages was undertaken to study the mechanism of action of cations and of heat-labile opsonin on engulfment. The rate of uptake of the particles was stimulated by Ca⁺⁺, Mg⁺⁺, Mn⁺⁺, or Co⁺⁺. At high concentrations (> 20 mM) Ca⁺⁺ and Mg⁺⁺ inhibited the rate of ingestion. Treatment of the particles with fresh serum (heat-labile opsonin) also stimulated the rate of ingestion. ¹²⁵I-labeled C3 was bound to the particles during opsonization. C3-deficient human serum lacked opsonic activity, which was restored by addition of purified C3. Normal, C2-deficient, and hereditary angioneurotic edema sera had equivalent opsonic activity. The serum opsonic activity thus involved C3 fixation to the particles by means of the properdin system. Although Mg⁺⁺ and heat-labile opsonin both accelerated the maximal rates of ingestion of the particles, neither altered the particle concentrations associated with one-half maximal ingestion rates. Opsonization of the particles markedly diminished the concentrations of divalent cations causing both stimulatory and inhibitory effects on ingestion rates and altered the shapes of the cation activation curves. ⁴⁵Ca was not bound to the particles during opsonization. The results are consistent with a mechanism whereby divalent cations and heat-labile opsonin activate ingestion by stimulating the work of engulfment rather than by merely enhancing cell-particle affinity, and whereby heat-labile opsonin acts by potentiating the effects of divalent cations.     

Studies on the Pentose Isomerases of Lactic Acid Bacteria

d-Xylose isomerase requires manganese ions for its action, but l-arabinose isomerase has a less specific on metal requirement. l-Arabinose isomerase is activated by addition of Mn⁺⁺ or Co⁺⁺, less effectively by addition of Zn⁺⁺, Ca⁺⁺, Mg⁺⁺, Sr⁺⁺ or Cd⁺⁺. Moreover, manganese and potassium ions for d-xylose isomerase, and manganese and cobaltous ions for l-arabinose isomerase were also shown to have protective effect on respective enzymes against thermal inactivation.     

On the selective adsorption of cations in the cell wall of the green algaValonia utricularis

The selective adsorption of the cations Na⁺, K⁺, Mg⁺⁺ and Ca⁺⁺ by the cell wall of the Mediterranean algaValonia utricularis (Siphonocladales, Chlorophyceae) from sea water of 40 %. S was investigated by extraction of cell-wall preparations, eluted before in 1.1 mol methanol (adjusted to pH 8) with 0.1 n formic acid in a Soxhlet apparatus. Na⁺ and K⁺ were determined by flame photometry, Mg⁺⁺ and Ca⁺⁺ by complexometric titration with EDTA. From calculation of the dry weight:fresh weight ratios and the chloride determinations in the eluates, the Donnan free-space fraction of the total cell-wall volume was calculated to about 35 %, and the analytical results of the cation concentrations in the extracts expressed asVal cm^–3 DFS. This calculation is based on the assumption that the acidic groups of the noncellulosic matrix material, carrying negative charges by dissociation at the reaction of sea water (ph about 8) are responsible for the adsorption of cations by exhibition of a Donnan effect. The results obtained show clearly that besides the divalent cations Mg⁺⁺ and Ca⁺⁺, which according to the physico-chemical laws of the Donnan distribution must be relatively accumulated to the second power of the monovalent ones, potassium is also enriched by selective adsorption, and the K⁺:Na⁺ ratio increased significantly compared with that in sea water. This seems to indicate that the strength of attraction between the cations and the negative sites is dependent on the radii of the ions and the state of hydration and/or polarisation of the ions and binding sites.     

Cation activation of desoxyribonuclease

The activation of desoxyribonuclease on desoxyribonucleate, known to occur with Mg⁺⁺ and Mn⁺⁺, has been shown to occur equally well with Co⁺⁺, to nearly the same extent with Fe⁺⁺, and to a lesser extent with Ca⁺⁺, Ba⁺⁺, Sr⁺⁺, Ni⁺⁺, Cd⁺⁺, and Zn⁺⁺. The conditions under which the optimal activation is revealed vary among these ions. Thus, Mg⁺⁺, Mn⁺⁺, and Co⁺⁺ may show marked activation under conditions in which Fe⁺⁺ is nearly ineffective. Since too high a concentration of an ion may be as ineffective as too little, concentration-activation curves were determined for each ion. Per micromole of nucleic acid phosphorus, the optimal effective amount of each ion in micromoles is as follows: Mg⁺⁺ 3, Mn⁺⁺ 3, Co⁺⁺ 3, Fe⁺⁺ 0.3, Ni⁺⁺ 0.3, Ba⁺⁺ 1.7, Ca⁺⁺ 3, Sr⁺⁺ 3, Zn⁺⁺ 0.3, and Cd⁺⁺ 0.3.The optimum pH for the activation with Mg⁺⁺, Co⁺⁺, and Ca⁺⁺ is about 6.5, that with Fe⁺⁺ is at 5.7, while Mn⁺⁺ shows two optima at pH 6.8 and 8.0.Experiments conducted in Pyrex and in quartz vessels showed the same results, and indicated that there was no activation of desoxy-ribonuclease in the absence of added salts.     

Bioelectric effects of ions microinjected into the giant axon of Loligo

1. A technique is described for recording the bioelectric activity of the squid giant axon during and following alteration of the internal axonal composition with respect to ions or other substances. 2. Experimental evidence indicates that the technique as described is capable of measuring changes in local bioelectric activity with an accuracy of 10 to 15 per cent or higher. 3. Alterations of the internal K⁺ or Cl^- concentrations do not cause the change in resting potential expected on the basis of a Donnan mechanism. 4. The general effect of microinjection of K⁺ Rb⁺, Na⁺, Li⁺, Ba⁺⁺, Ca⁺⁺, Mg⁺⁺, or Sr⁺⁺ is to cause decrease in spike amplitude, followed by propagation block. 5. The resting potential decreases when the amplitude of the spike becomes low and block is incipient. 6. The decrease in resting potential and spike amplitude may be confined to the immediate vicinity of the injection. 7. At block, the resting potential decreases up to 50 per cent, but injection of small quantities of divalent cations may cause much larger localized depolarization. 8. The blocking effectiveness of K⁺, Na⁺, and Ca⁺⁺ expressed as reciprocals of the relative amounts needed to cause block is approximately 1:5:100. Rb⁺ has the same low effectiveness as does K⁺. Li⁺ resembles Na⁺. Ba⁺⁺ and Mg⁺⁺ are approximately as effective as Ca⁺⁺. 9. Microinjection of Na⁺ may cause marked prolongation of the spike at the injection site as well as decrease in its amplitude. 10. The anions used (Cl^-, HCO₃^-, NO₃^-, SO₄^-, aspartate, and glutamate) do not seem to exert specific effects. 11. A tentative explanation is offered for the insensitivity of the resting potential to changes in the axonal ionic composition. 12. New data are presented on the range of variation, in a large sample, of the magnitude of the resting potential and spike amplitude.     

3H-mepyramine binding to histamine H1-receptors in guinea pig lung: Characteristics and regulation by ions

The antogonist [³H]-mepyramine is used to label histamine H₁-receptors in guinea pig lung. Scatchard analysis reveals two classes of binding sites. Monovalent cations decrease steady-state binding (Na⁺ > Li⁺ > K⁺), while divalent cations (Mg⁺⁺, Ca⁺⁺, Mn⁺⁺, Ba⁺⁺) exhibit a biphasic curve, increasing binding at low concentrations and decreasing it at higher levels. Na⁺ decreases both affinity and number of binding sites. Dissociation curve shows two components, and Na⁺ accelerates the rate of dissociation of the slower component. GTP does not affect the binding of the antagonist ³H-Mepyramine.     

Histochemical study of TPPase and NDPase in the human nervous tissue,with particular reference to glia cells

Summary TPPase and IDPase activities were investigated in normal and reactive human nervous tissue with three divalent ions as activators at different concentrations. Enzyme distribution in neuronal Golgi apparatus, vessels and glia cells is described. In glia cells IDP was hydrolyzed almost equally with the different ions at the concentration of 0.005 M, while TPP was only weakly hydrolyzed with Ca⁺⁺ and Mg⁺⁺. Small positive structures in the cells remained positive and for these a correspondence with the Golgi apparatus is suggested.Partially supported by Consiglio Nazionale delle Ricerche (C.N.R), Rome.