CALCULATIONS OF BIOELECTRIC POTENTIALS : IV. SOME EFFECTS OF CALCIUM ON POTENTIALS IN NITELLA

In Nitella the substitution of KCl for NaCl changes the P.D. in a negative direction. In some cases this change is lessened by adding solid CaCl₂ to the solution of KCl. This may be due to lessening the partition coefficient of KCl or to decreasing the solubility of an organic substance which sensitizes the cell to the action of KCl. Little or no correlation exists between this effect of calcium and its ordinary antagonistic action in producing a balanced solution which preserves the life of the cell indefinitely. CaCl₂ is negative to NaCl but positive to KCl. The effects of mixtures of KCl, NaCl, and CaCl₂ are discussed. The concentration effect of a mixture of KCl + CaCl₂ shows certain peculiarities due to action currents: these resemble those found with pure KCl. These studies and others on Nitella, Valonia, and Halicystis indicate that mobilities and partition coefficients are variable and can be brought under experimental control.     

A relationship between 13C-chemical-shift displacements and counterion-condensation theory, in the binding of calcium ion by heparin

Characteristics of the interaction between heparin and calcium ion in the presence of sodium ion have been examined by monitoring the 13C-chemical shift changes as a function of the calcium ion concentration and the total ionic strength. The results indicated that the association between the polyanion and the divalent cation is a delocalized process, as opposed to one involving specific binding. The correspondence found between chemical shift and the number of Ca2+ ions bound per charged group, as derived from the Manning counterion-condensation model, showed that the stoichiometry is not a constant quantity but, rather, varies throughout the titration, and approaches a limiting value of 2 at high dilution. Additional measurements of T1 and line-width were consistent with an intramolecular order-disorder conformational process induced by the binding of calcium ion. Moreover, binding does not occur or is relatively weak with N-desulfated heparin, or chondroitin 4-sulfate and 6-sulfate, each of which possesses fewer sulfate groups than heparin. These differences serve to emphasize the importance of the charge-density parameter in the control of counterion condensation according to the Manning model, and suggest that the spacing between the negatively charged groups is an associated factor.     

The Thermodynamic Activity of Calcium Ion in Sodium Chloride-Calcium Chloride Electrolytes

Experimental data on the mean activity coefficient of CaCl₂ in NaCl-CaCl₂ mixtures at ionic strengths below 1 m have been used to prepare a table of activity coefficients for Ca⁺⁺ in solutions of physiological interest. The establishment of an empirical calcium ion activity scale is discussed, and a number of possible assumptions are examined. The assumption γ++ = (γ±)² is suggested as being the simplest with a theoretical basis.     

The effect of metal ions on the amidolytic activity of human factor Xa (Activated Stuart-Prower Factor)

The effect of metal ions on human activated Factor X (Factor X_a) hydrolysis of the chromogenic substrate benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222) was studied utilizing initial rate enzyme kinetics. The divalent metal ions Ca²⁺, Mn²⁺, and Mg²⁺ enhanced Factor X_a amidolytic activity with K_m values of 30 μm, 20 μm, and 1.4 mm, respectively. Na⁺ activation of Factor X_a amidolytic activity was also found. The K_m for Na⁺ activation was 0.31 m. Both the divalent metal ions and Na⁺ increased the affinity of Factor X_a for S2222 and had no effect on the maximal velocity of the reaction. Other monovalent cations were unable to activate Factor X_a. However, K⁺ was a competitive inhibitor of the Na⁺ activation (K_i = 0.14 m). Lanthanide ions inhibited Factor X_a amidolytic activity. Gd³⁺ inhibition of Factor X_a hydrolysis of S2222 was noncompetitive and had a K_i of 3 μm. The lanthanide ion inhibition could not be reversed by Ca²⁺ even when Ca²⁺ was present in a 1000-fold excess over its K_m indicating nonidentity of the Factor X_a lanthanide and Ca²⁺ binding sites. It is concluded that the Factor X_a Ca²⁺ binding sites have characteristics different from those previously described for the Factor X molecule and that Mg²⁺, Na⁺, and K⁺ may be physiological regulators of Factor X_a activity.     

Accommodation of Ca(II) ions for catalytic activity by a group I ribozyme

The wildtype Tetrahymena ribozyme cannot catalyze detectable levels of phosphotransfer activity in vitro on an exogenous RNA substrate oligonucleotide when calcium(II) is supplied as the only available divalent ion. Nevertheless, low-error mutants of this ribozyme have been acquired through directed evolution that do have activity in 10 mM CaCl₂. The mechanisms for such Ca(II) accommodation are not known. Here, we assayed the entire molecule in an effort to identify the roles of the mutations in allowing catalytic activity in Ca(II). We used four biochemical probing techniques - native-gel electrophoresis, hydroxyl radical footprinting, terbium(III) cleavage footprinting, and phosphorothioate interference mapping - to compare the solution structure of the wildtype ribozyme with that of a Ca(II)-active five-site mutant. We compared the gross folding patterns and specific metal-binding sites in both MgCl₂ and CaCl₂ solutions. We detected no large-scale folding differences between the two RNAs in either metal. However, we did discover a limited number of local folding differences, involving regions of the RNA affected by positions 42, 188, and 270. These data support the notion that Ca(II) is accommodated by the Tetrahymena ribozyme by a slight breathing at the active site, but that alterations at, near to, and distal from the active site can all contribute to Ca(II)-based activity.     

Interaction of Heparins and Dextran Sulfates with a Mesoscopic Protein Nanopore

A mechanism of how polyanions influence the channel formed by Staphylococcus aureus α-hemolysin is described. We demonstrate that the probability of several types of polyanions to block the ion channel depends on the presence of divalent cations and the polyanion molecular weight and concentration. For heparins, a 10-fold increase in molecular weight decreases the half-maximal inhibitory concentration, IC₅₀, nearly 10⁴-fold. Dextran sulfates were less effective at blocking the channel. The polyanions are significantly more effective at reducing the conductance when added to the trans side of this channel. Lastly, the effectiveness of heparins on the channel conductance correlated with their influence on the ζ-potential of liposomes. A model that includes the binding of polyanions to the channel-membrane complex via Ca²⁺-bridges and the asymmetry of the channel structure describes the data adequately. Analysis of the single channel current noise of wild-type and site-directed mutant versions of α-hemolysin channels suggests that a single polyanion enters the pore due to electrostatic forces and physically blocks the ion conduction path. The results might be of interest for pharmacology, biomedicine, and research aiming to design mesoscopic pore blockers.     

EFFECTS OF pH AND OF VARIOUS CONCENTRATIONS OF SODIUM,POTASSIUM, AND CALCIUM CHLORIDE ON MUSCULAR ACTIVITY OF THE ISOLATED CROP OF PERIPLANETA AMERICANA (ORTHOPTERA)

1. Twenty-five solutions which contained KCl (0.0, 0.2, 0.4, 0.6, and 0.8 gm. per liter), in combination with CaCl₂ (0.0, 0.2, 0.4, 0.6, and 0.8 gm. per liter), 10.0 gm. of NaCl, and 0.2 gm. of NaHCO₃ per liter of solution were tested in order to determine satisfactory KCl/CaCl₂ ratios in an insect physiological salt mixture for the maintenance of muscular activity by the isolated crop of the American roach. Satisfactory activity products (0.390 to 0.549) were obtained in seven mixtures with KCl/CaCl₂ ratios of 0.2/0.2, 0.4/0.4, 0.6/0.6, 0.8/0.8, 0.2/0.4, 0.4/0.6, and 0.6/0.8, expressed as gram per liter. These ratios lie between 0.50 and 1.00. In solutions which contained calcium, but no potassium, approximately 50 per cent of the crops exhibited an initial tone increase and were arrested in rigor. See Fig. 2. In solutions which contained potassium, but no calcium, all crops showed an initial loss of tone and arrest in relaxation. See Fig. 2. 2. Seven KCl/CaCl₂ ratios (see paragraph 1 above) were tested with eight NaCl concentrations (1.0, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, and 1.8 per cent) at a pH of 8.0. In these mixtures, the ones with KCl/CaCl₂ ratios of less than 1.0 produced higher activity products than those with ratios equal to 1.00. The highest average activity product (0.849) was obtained in the solutions with 0.2 gm. of KCl and 0.4 gm. of CaCl₂ per liter. 3. Four KCl/CaCl₂ ratios (0.2/0.2, 0.4/0.4, 0.2/0.4, and 0.4/0.6 gm. per liter) were tested with 1.4, 1.5, and 1.6 per cent NaCl at a pH of 7.5. When analyzed with data from comparable solutions at a pH of 8.0, it was found that 1.4 per cent NaCl afforded an optimum environment for isolated crop activity. 4. Effects of hydrogen and hydroxyl ion concentrations were studied at pH values of 6.8, 7.5, 8.0, and 8.9. The highest average activity product, 1.011, was produced at a pH of about 8.0. 5. A satisfactory physiological salt solution for the isolated foregut of the American roach, Periplaneta americana, would contain 14.0 gm. of NaCl, 0.4 gm. of CaCl₂, 0.2 gm. of KCl, and 0.2 gm. of NaHCO₃ per liter of solution. This mixture should have a pH value between 7.8 and 8.2. 6. Durations of crop activity extending over periods as long as 25 hours were quite common, and several crops maintained contractions for more than 30 hours. The greatest longevity was for crop 814, from a female, which continued activity for slightly more than 47 hours. 7. A significant difference between the activity products of the crops from males and the crops from females was recorded. Although there was not a significant difference in the amount of food ingested by males and females, 12 hours after feeding there was more food in the females'' crops, and the food progressed more rapidly through the males'' crops than through the females''. In addition, crops from the two sexes reacted differently to the effects of day old solutions. This sex difference is apparently related to an inherently increased activity of the crop from the male roach.     

Calcium effect on the mycelial cell walls of Botrytis cinerea

The goal of this study was to determine whether calcium ion, (one of the electrolytes released after plant cell attack), may have a direct effect on fungal growth and chemistry of the fungal cell wall. B. cinerea was grown on Richard's solution containing different amounts of CaCl₂, and the cell walls were extracted from the mycelium after 7 days of growth. Mineral, neutral and aminosugar, protein and uronic acid contents were determined. At 1 g l⁻¹ CaCl₂, only the aminosugar content increased. At 2 g l⁻¹ CaCl₂, neutral sugar synthesis was reduced, whereas the uronic acid content increased. For higher CaCl₂ concentrations, the calcium ion content of the cell wall increased, resulting in reduced protein and neutral sugar contents. Meanwhile, the cell wall proportion of the mycelia increased on a dry weight basis due to an increase in uronic acid, Ca, P, Na and neutral sugar contents of the cell wall with increasing CaCl₂ in the media. The resulting thickening of the fungal cell wall caused by calcium ion may be an important factor in the host-pathogen relationship.     

Mean activity coefficients for the simple electrolyte in aqueous mixtures of polyelectrolyte and simple electrolyte. V. common counterion mixtures of alkali-metal dextransulfates with alkali metal chlorides

Mean molal activity coefficients of simple electrolyte in aqueous solutions of Li, Na, K or Cs salts of dextransulfate (DS) with added LjCl, NaCl, KCl or CsCl are reported. The measurements were carried out by means of an electrochemical cell method using a cation exchange membrane as cation selective electrode and Ag/AgCl electrodes. For LiDS-LiCl, NaDS-NaCl and CsDS-CsCl systems the polymer concentration, m_p, was varied from 0.0088 to 0.113 m and at a given m_p the ratio X of the polymer to salt concentration was varied from 0.5 to 16. Due to the insolubility of KDS in high concentration of KCl, the measurements on KDS-KCl system were performed in the m_p range of 0.0088–0.089 m and some of the smaller X values were omitted. The activity coefficient results are compared to Manning's limiting laws, the additivity rule, and to new limiting laws. The additivity rule can give an excellent representation of the data for all m_p values when γ_p is used as an adjustable parameter.     

The binding of divalent metal ions to polyelectrolytes in mixed counterion systems. I. The dye spectrophotometric method

The dye spectrophotometric method for the measurement of the activity of divalent metal ions in polyelectrolyte solutions containing added electrolytes is discussed.The method is applied to mixtures containing the dextransulfate polyanion, NaCl, and MgCl2 or Ca2. A two wavelength ratio method as applied to polyelectrolyte solutions is compared to the standard method which makes use of the previous determination of the dye-metal ion formation constant. The ratio method is found to be a convenient and reliable method which is not influenced by decomposition of the dye or by statistical errors in the extrapolation procedure. The activity coefficients as determined by the two wavelength dye spectrophotometric method are compared to results of Donnan exclusion measurements, and of EMF measurements using a calcium ion selective electrode. The results of the spectrophotometric method are equal to those of the two other methods within the limits of error in the latter. The spectrophotometric measurements can extend to much lower ion activaties than the other two methods, and can be done in the presence of a large excess of added electrolyte, yielding results of considerably improved precision when compared to Donnan and EMF methods.     

Calcium Ion-Dependent Increase in Thermostability of Dextran Glucosidase from Streptococcus mutans

Dextran glucosidase from Streptococcus mutans (SmDG), which belongs to glycoside hydrolase family 13 (GH13), hydrolyzes the non-reducing terminal glucosidic linkage of isomaltooligosaccharides and dextran. Thermal deactivation of SmDG did not follow the single exponential decay but rather the two-step irreversible deactivation model, which involves an active intermediate having 39% specific activity. The presence of a low concentration of CaCl₂ increased the thermostability of SmDG, mainly due to a marked reduction in the rate constant of deactivation of the intermediate. The addition of MgCl₂ also enhanced thermostability, while KCl and NaCl were not effective. Therefore, divalent cations, particularly Ca²⁺, were considered to stabilize SmDG. On the other hand, CaCl₂ had no significant effect on catalytic reaction. The enhanced stability by Ca²⁺ was probably related to calcium binding in the β→α loop 1 of the (β?α)₈ barrel of SmDG. Because similar structures and sequences are widespread in GH13, these GH13 enzymes might have been stabilized by calcium ions.     

A proposed mechanism for the thermal denaturation of a recombinant Bacillus halmapalus α-amylase—the effect of calcium ions

The thermal stability of a recombinant α-amylase from Bacillus halmapalus α-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). This α-amylase is homologous to other Bacillus α-amylases where crystallographic studies have identified the existence of three calcium binding sites in the structure. Denaturation of BHA is irreversible with a T_m of approximately 89 °C and DSC thermograms can be described using a one-step irreversible model. A 5 °C increase in T_m in the presence of 10-fold excess CaCl₂ was observed. However, a concomitant increase in the tendency to aggregate was also observed. The presence of 30–40-fold excess calcium chelator (ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis[β-aminoethyl ether] N,N,N′,N′-tetraacetic acid (EGTA)) results in a large destabilization of BHA, corresponding to about 40 °C lower T_m as determined by both CD and DSC. Ten-fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which probably originate from different populations of BHA/calcium complexes. Combined interpretation of these observations and structural information on homologous α-amylases forms the basis for a suggested mechanism underlying the inactivation mechanism of BHA. The mechanism includes irreversible thermal denaturation of different BHA/calcium complexes and the calcium binding equilibria. Furthermore, the model accounts for a temperature-induced reversible structural change associated with calcium binding.     

Effect of calcium phosphate and alumina C γ gels on the mutagenicity and cytotoxicity of dimethylnitrosamine as studied in the CHO/HGPRT system

When CaCl₂ was added in increasing concentrations to a rat liver metabolic activation system (S9) buffered with sodium phosphate, the mutagenic activity and cytotoxicity of dimethylnitrosamine (DMN) in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system were greatly increased. This effect was not observed with an S9 mix buffered with N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES). The calcium phosphate gel precipitate of the S9 mix possessed approximately $\text{built1}\text{3}$ of the total activity of the mix, while the supernatant had only slight activity. However, when the calcium phosphate gel precipitate of a solution of S9 salts (without S9 protein) was added to the supernatant, the remaining $\text{2}\text{3}$ of the activity was recovered. Commercially obtained calcium phosphate, tricalcium phosphate, and alumina C γ gels could substitute for CaCl₂ in the S9 mix, but diethylaminoethyl cellulose (DEAE cellulose) could not. Alumina C γ gel can exert its effect in the absence of both CaCl₂ and phosphate in the S9 mix. Increasing the time of contact between the S9 protein and the S9 salts increased the efficacy with which the S9 mix activated DMN; this is indicative of an adsorptive process by calcium phosphate gel.     

Alleviation of NaCl toxicity in the cyanobacterium <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7942 by exogenous calcium supplementation

Salinity (NaCl) is one of the major problems associated with irrigated agricultural lands, especially rice fields. Being the common inhabitants of rice fields, cyanobacteria frequently experience high concentration of NaCl which in turn causes cellular damage. Therefore, mitigation of NaCl stress in cyanobacteria, plant growth-promoting microorganisms, is of utmost importance. The present study was designed to investigate the role of calcium in the alleviation of NaCl stress-induced cellular in Synechococcus sp. PCC 7942. The cyanobacterium was subjected to sub-lethal concentration of NaCl (800 mM) with and without the supplementation of calcium (1 mM CaCl₂) for 8 days. The results showed a drastic reduction in growth due to excess NaCl, but supplementation of CaCl₂ reduced the salt stress damage and partially restored growth. Application of calcium increased pigment contents, photosynthetic efficiency, antioxidative enzyme activity, osmolyte contents and reduced the intracellular sodium ion concentration, MDA content, electrolyte leakage and free oxygen radical generation. Furthermore, proteins involved in photosynthesis, respiration, ATP synthesis and protein synthesis along with two hypothetical proteins were also observed to be upregulated in the cyanobacterium in presence of calcium. Furthermore, proteins related to oxidative stress defence, nitrogen metabolism, carbohydrate metabolism, fatty acid metabolism and secondary metabolism were found to be upregulated by several fold. Therefore, our study suggests that calcium suppresses salt toxicity in Synechococcus sp. PCC 7942 by restricting the entry of Na⁺ into the cell, increasing osmolyte production and upregulating defence-related proteins.     

Mg and Ca binding to heparin in the presence of added univalent salt

Activities of Mg and Ca in solutions of heparin with added sodium or potassium chloride have been determined by means of a previously described dye spectrophotometric method. The polyion concentration, C_p (expressed as mol univalent anionic charges/I) is 0.001 in all cases, solution total ionic strengths are 0.005, 0.0075, 0.01, 0.02 and 0.04 mol/l. Divalent metal ion concentrations are varied between 0 and 1.0 C_p. The results for the metal ion activities are expressed in the form of binding isotherms, θ₂ vs. C₂/C_p (θ₂=C_2b/C_p; C_2b=bound divalent metal ion concentration) and Scatchard plots, K₂ vs. θ₂/(C₂-C_2b, at different ionic strengths. Results are correlated with a theoretical treatment of the mixed counterion system, called the ‘two variable theory’, recently developed by Manning. The ionic strength dependence of θ₂ and K₂ is found to agree with the prediction of the two variable theory. Mg interaction with heparin appears to be independent of the nature of the charged groups on the polyion and is in very good agreement with the two variable theory. Ca binding was found to be considerably higher than Mg binding and is in excess of theoretical predictions, suggesting a localized or specific interaction of Ca with heparin.     

An alternative explanation for phase transitions observed in quick frozen calcium cardiolipin solution

It is demonstrated that the phase changes reported for a quick frozen calcium cardiolipin solution containing CaCl₂ are virtually identical to those seen in pure CaCl₂. This introduces uncertainty as to whether the data in fact reflect the behavior of cardiolipin or of the associated CaCl₂.     

The Adsorption of Bacteriophage ϕX174 to its Host

The adsorption of purified ϕX174 to E. coli C and to E. coli C cell walls was investigated. Adsorption was measured by assaying for unadsorbed plaque formers. The amount of irreversible and reversible adsorption depends upon pH and divalent ion concentration. Maximum irreversible adsorption occurs in 0.1 M CaCl₂ at 36°C. There is no detectable reversible adsorption at conditions of pH and CaCl₂ concentration optimum for irreversible adsorption. Under these optimum conditions, diffusion is not the rate-limiting factor, and the encounter efficiency appears to be low. The rate constant is 1.0 × 10^-10 ml/sec. Phages adsorbed irreversibly to live cells cause infection and to the isolated cell walls apparently cause release of DNA. There is a specific ϕX174 receptor site on the mucocomplex portion of the cell wall.     

Saline groundwater as an aquaculture medium: physiological studies on the red drum, Sciaenops ocellatus

Physiological responses of the euryhaline red drum, Sciaenops ocellatus, to chloride salt addition, low salinity, and high sulfate concentration were measured. Survival was increased by addition of calcium chloride (CaCl₂) or magnesium chloride (MgCl₂) to dilute artificial seawater (0.2 ppt salinity). Although survival and routine metabolic rates were greater in MgCl₂ treatments, growth and feed efficiency were greater in CaCl₂ treatments. Marginal metabolic scope increased when CaCl₂ or MgCl₂ were added to dilute artificial seawater. There was a strong positive linear relationship (p=0.0001, r=0.91) between fish survival and salinity of artificial seawater dilutions over the salinity range 0.1 to 3.0 ppt. Monovalent ion concentrations in red drum plasma varied; whereas, divalent ion concentrations were relatively constant. Survival and growth were not affected by high sulfate concentrations (2000 mg l^-1) in 3.0 ppt artificial seawater supplemented with either sodium sulfate or magnesium sulfate. Routine metabolic rate and marginal metabolic scope of red drum exposed to high sulfate concentrations were slightly, but not significantly, lower than those of red drum in 3 ppt artificial seawater.