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.     

Acid Metallophosphatase from the Ameba Amoeba proteus

Tartrate-resistant acid phosphatase (TR-AcPh) from the ameba Amoeba proteus is represented by 3 bands (electromorphs) revealed after disk-electrophoresis in PAAG, using 2-naphthylphosphate as substrate. The presence of 50 mmol/l MgCl₂ or CaCl₂ in the incubation mixture increases activities of all electromorphs of TR-AcPh, while of ZnCl₂, of two of them. The activity of the TR-AcPh electromorphs also rose after the 30-min incubation of the gels in MgCl₂, CaCl₂ or ZnCl₂ (10 and 100 mM) before gel staining. However, 1 M ZnCl₂, unlike 1 M CaCl₂ or 1 M MgCl₂, partly inactivated two out of three TR-AcPh electromorphs. The TR-AcPh electromorphs were inhibited by 1,10-phenanthroline (1,10-Ph), EDTA, and EGTA (all at a concentration of 5 mM) faster than by H₂O₂ (10 mM). The inactivation of the TR-AcPh electromorphs by the chelating agents did not depend (EGTA) or nearly did not depend (EDTA, 1,10-Ph) on their concentration (0.05, 0.5, and 5 mM). Out of 5 tested ions (Mg²⁺, Ca²⁺, Fe²⁺, Fe³⁺, and Zn²⁺), only Zn ions reactivated the TR-AcPh electromorphs inactivated by 1,10-Ph, EDTA or EGTA. The TR-AcPh electromorphs were reactivated worse after inactivation by EGTA than by EDTA or 1,10-Ph. It is suggested that the active site of TR-AcPh contains the zinc ion essential for catalytic activity of this enzyme, i.e., TR-AcPh of A. proteus is a metallophosphatase performing the phosphomonoesterase activity in acidic medium.     

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.     

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.     

Effects of ionic concentration and pH upon the state of aggregation of Neurospora mitochondrial malate dehydrogenase

Five active isoenzymes of $\text{Neurospora}$ mitochondrial malate dehydrogenase of 105,000, 91,000, 78,000, 65,000 and 39,000 daltons were observed when the enzyme was extracted from mycelia and centrifuged in a sucrose gradient with 5 mM tris-Cl at pH 9. Only one active species of 65,000 daltons was observed in either 100 mM tris-Cl, pH 9, or 5 or 100 mM sodium citrate at either pH 4 or 6. The addition of 10 mM of either MgCl₂ or CaCl₂ to the 5 mM tris-Cl pH 9 buffer reversed the aggregation and led to the occurrence of only the 65,000 daltons species. The addition of either 10, 50 or 100 mM KCl to the 5 mM tris-Cl pH 9 buffer yielded 4, 3 and 1 isoenzymes, respectively. The latter's molecular weight was 65,000. Thus, in alkaline solution, monovalent cations at 100 mM and divalent cations at 10 mM prevent the formation of multiple molecular weight species.     

A requirement for chelation in obtaining functional chloroplasts of sunflower and wheat.

In studying conditions for obtaining photosynthetically functional chloroplasts from mesophyll protoplasts of sunflower and wheat, a strong requirement for chelation was found. The concentration of chelator, either EDTA or pyrophosphate (PP_i), required for maximum activation depended on the pH, the concentration of orthophosphate (P_i) in the assay, and the chelator used. Studies with EDTA indicate that including the chelator in the isolation, resuspension, and assay media, in the absence of divalent cations, was most effective. Increased concentration of EDTA from 1 to 10 mm broadened the pH response curve for photosynthesis, inasmuch as a higher concentration of chelator was required for activation of photosynthesis at lower pH.Either EDTA, PP_i, or citrate could activate photosynthesis of sunflower chloroplasts isolated and assayed at pH 8.4. At pH 7.6, PP_i and EDTA were equally effective at low P_i concentrations but PP_i was particularly effective in shortening the induction period at high concentrations of P_i (2.5 mm) in the assay medium. Including 1 mm 3-phosphoglycerate in the assay medium with or without P_i could not replace the need for chelation. However, 3-phosphoglycerate + EDTA in the assay medium with 0.5 mm P_i, pH 7.6, gave a short induction period and rates of photosynthesis similar to those with 10 mm PP_i. The results suggest that PP_i can have a dual effect at the lower pH through chelation and inhibition of the phosphate transporter.Photosynthesis by sunflower chloroplasts isolated and assayed at pH 8.4 with 0.2 mm EDTA (+ 0.5 mm P_i in the assays) was severely inhibited by 2 mM CaCl₂, MgCl₂, or MnCl₂. Wheat chloroplasts isolated and assayed at pH 8.4 without chelation, and assayed with 0.2 mm P_i, had low rates of photosynthesis (25 μmol O₂ evolved mg^?1 chlorophyll h^?1) which were strongly inhibited by 2 to 4 mm MgCl₂, MnCl₂, or CaCl₂. With inclusion of EDTA and P_i at optimum levels, isolated chloroplasts of sunflower and wheat have high rates of photosynthesis and PP_i or divalent cations are not of benefit.     

Calcium-induced transformation of cardiolipin nanodisks

Miniature membranes comprised of tetramyristoylcardiolipin (CL) and apolipoprotein (apo) A-I, termed nanodisks (ND), are stable, aqueous soluble, reconstituted high density lipoproteins. When CL ND, but not dimyristoylphosphatidylcholine (PC) ND, were incubated with CaCl₂, a concentration dependent increase in sample turbidity occurred, consistent with CL undergoing a bilayer to non-bilayer transition. To assess the cation specificity of this reaction, CL ND were incubated with various mono- and divalent cations. Whereas monovalent cations had no discernable effect, MgCl₂ and SrCl₂ induced a response similar to CaCl₂. When ND were formulated using different weight ratios of CL and PC, those possessing 100% CL or 75% CL remained susceptible to CaCl₂ induced sample turbidity development while ND possessing 50% CL displayed reduced susceptibility. ND comprised of 25% CL and 75% PC were unaffected by CaCl₂ under these conditions. SDS PAGE analysis of insoluble material generated by incubation of CL ND with CaCl₂ revealed that nearly all apoA-I was recovered in the insoluble fraction along with CL. One h after addition of EDTA to CaCl₂-treated CL ND, sample clarity was restored. Collectively, the data are consistent with a model wherein Ca²⁺ forms a bidentate interaction with anionic phosphates in the polar head group of CL. As phosphate group repositioning occurs to maximize Ca²⁺ binding, CL acyl chains reposition, accentuating the conical shape of CL to an extent that is incompatible with the ND bilayer structure.     

Structural changes in lecithin-water systems thermal-turbidimetric studies

Turbidity measurements were made of dilute aqueous dispersions of 1,2-dipalmitoyl-l-lecithin as the temperature was varied. In the range from 24 to 33° a decrease in turbidity is associated with the penetration of water between the layers of lipid in the crystalline structure. At the transition to the liquid crystalline form (39–41°) a sharp decrease in turbidity occurs. In solutions of 0.06 M LiCl, 1 mM CaCl₂, MgCl₂ or 0.05 M PO₄³ an increase in turbidity resulted at the transition temperature, whereas in 0.1 M NaCl, or quarternary ammonium salts the turbidity decreased. The presence of small amounts of dicetylphosphoric acid mixed with the lecithin decreased the turbidity, and at elevated pH levels there was no change at the transition temperature.     

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.     

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.     

Electrophysiological responses to salts from antennal chaetoid taste sensilla of the ground beetle Pterostichus aethiops

Antennal gustatory sensilla of the ground beetle Pterostichus aethiops (Pz., 1797) (Coleoptera, Carabidae) respond to salts, the three sensory cells, A-, B- and C-cells, producing action potentials that are distinguished by differences in their shape, amplitude, duration and polarity of spikes. The B-cell (salt cell) was highly sensitive to both ionic composition and concentration of the tested nine salt solutions showing phasic-tonic type of reaction with a pronounced phasic component. The stimulating effect was dominated by the cations involved, and in most cases, monovalent cations were more effective stimuli than divalent cations. Salt concentration/response relations were tested with NaCl at 1, 10, 100 and 1000 mmol l⁻¹: mean firing rates increased from 0.8 to 44 spikes per first second of the response, respectively. The pH value of the stimulating solutions also influenced the B-cell rate of firing. By contrast, the pH level of stimulus solutions influenced the A-cells’ phasic-tonic response more than the ionic composition or concentration of these solutions. Compared to a standard 100 mmol l⁻¹ salt (NaCl) solution (pH 6.3), alkaline solutions of the salts NaCH₃COO, Na₂HPO₄ and Na₂B₄O₇ (pH 7.9, 8.5 and 9.3, respectively, all 100 mmol l⁻¹) induced remarkably stronger responses in the A-cell. On the other hand, the reaction to an acid solution of NaH₂PO₄ (pH 4.5, 100 mmol l⁻¹) was minimal. A-cell responses to neutral salts like NaCl, KCl, CaCl₂, MgCl₂ and C₅H₁₄NOCl (pH 6.1-6.5) varied largely in strength. Very low or no responses were observed with chlorides of divalent cations, CaCl₂ and MgCl₂, and choline chloride (C₅H₁₄NOCl), indicating that the ionic composition of the solutions also affected A-cell responses. Neural activity of the C-cell was not influenced by the salt solutions tested.     

Isolation and purification of calcium and magnesium dependent endonuclease from rat liver nuclei

An endonuclease associated with rat liver chromatin was extracted with 0.6 M NaCl and purified by ammonium sulfate fractionation and Sephadex G-100 gel filtration. The enzyme produces single strand scissions on native DNA at neutral pH in the presence of 1 mM CaCl₂ and 5 mM MgCl₂. Alkali-denatured DNA was not nicked by the enzyme. Omission of Ca²⁺ reduced the enzyme activity to about one seventh. Without Ca²⁺, however, Mn²⁺ was more effective than Mg²⁺. The molecular weight of the enzyme is about 27,000.     

Correlation between monovalent cation-induced decreases in chlorophyll a fluorescence and chloroplast structural changes

Low concentrations (~ 3 mm) of salts of monovalent cations such as Na⁺, K⁺, and tetraethylammonium were found to decrease the turbidity of chloroplast suspensions. The turbidity changes (Δ₅₄₀) had the same kinetics, salt concentration dependence, and pH dependence as the monovalent cation-induced decreases in chlorophyll a fluorescence (9), suggesting that structural changes are the cause of the associated increases in spillover. Electron microscopy revealed that the grana are stacked when spillover is inhibited (in the absence of salts or the presence of divalent cations) and that monovalent cations cause the grana to unstack, thereby promoting spillover.     

Protection of Pyruvate,Pi Dikinase from Maize against Cold Lability by Compatible Solutes

Most C₄ species are chilling sensitive and certain enzymes like pyruvate,Pi dikinase of the C₄ pathway are also cold labile. The ability of cations and compatible solutes to protect maize (Zea mays) dikinase against cold lability was examined. The enzyme in desalted extracts at pH 8 from preilluminated leaves could be protected against cold lability (at 0°C) by the divalent cations Mn²⁺, Mg²⁺, and Ca²⁺. There was substantial protection by sulfate based salts but little protection by chloride based salts of potassium or ammonium (concentration 250 millimolar). The degree of protection against cold lability under limiting MgCl₂ (5 millimolar) was pH sensitive (maximum protection at pH 8), but independent of ionic strength (up to 250 millimolar by addition of KCl). In catalysis Mg²⁺ is required and Mn²⁺ could not substitute as a cofactor. Several compatible solutes reduced or prevented the cold inactivation of dikinase (in desalted extracts and the partially purified enzyme), including glycerol, proline, glycinebetaine and trimethylamine-N-oxide (TMAO). TMAO and Mg²⁺ had an additive effect in protecting dikinase against cold inactivation. TMAO could largely substitute for the divalent cation and addition of TMAO during cold treatment prevented further inactivation. Cold inactivation was partially reversed by incubation at room temperature; with addition of TMAO reversal was complete. The temperature dependence of inactivation at pH 8 and 3 millimolar MgCl₂ was evaluated by incubation at 2 to 17°C for 45 minutes, followed by assay at room temperature. At preincubation temperatures below 11°C there was a progressive inactivation which could be prevented by TMAO (450 millimolar). The results are discussed relative to possible effects of the solutes on the quaternary structure of this enzyme, which is known to dissociate at low temperatures.     

Reversible changes of canavalin solubility controlled by divalent cation concentration in crude sword bean extract

Canavalin is a vicilin-class (7S) storage protein found in sword bean (Canavalia gladiata). Our previous report indicated that canavalin is precipitated by the addition of 20 mM MgCl₂ to crude sword bean extract. Here, we examined the solubility changes induced by the addition of Mg²⁺ and Ca²⁺ at various concentrations. Canavalin tended to be insolubilized at relatively low concentrations of MgCl₂ (< 20 mM) and solubilized at relatively high concentrations (> 20 mM). In addition, canavalin was slightly insolubilized in the presence of NaCl. Overall, the results revealed that solubility changes are reversible and depend on the concentration of divalent cations. Therefore, we suggested a reaction scheme that describes the effects of divalent cations on the solubility of canavalin, which would facilitate the study of its physiological function and the application of canavalin in the food processing industry.     

Cation-Dependent Binding of Substrate to the Folate Transport Protein of Lactobacillus casei

Lactobacillus casei cells grown in the presence of limiting folate contained large amounts of a membrane-associated binding protein which mediates folate transport. Binding to this protein at 4°C was time and concentration dependent and at low levels (1 to 10 nM) of folate required 60 min to reach a steady state. The apparent dissociation constant (K_d) for folate was 1.2 nM at pH 7.5 in 100 mM K-phosphate buffer, and it varied by less than twofold when measured over a range of pH values (5.5 to 7.5) or in buffered salt solutions of differing ionic compositions. Conversely, removal of ions and their replacement with isotonic sucrose (pH 7.5) led to a 200-fold reduction in binding affinity for folate. Restoration of the high-affinity state of the binding protein could be achieved by the readdition of various cations to the sucrose medium. K_d measurements over a range of cation concentrations revealed that a half-maximal restoration of binding affinity was obtained with relatively low levels (10 to 50 μM) of divalent cations (e.g., Ca²⁺, Mg²⁺, and ethylenediammonium²⁺ ions). Monovalent cations (e.g., Na⁺, K⁺, and Tris⁺) were also effective, but only at concentrations in the millimolar range. The K_d for folate reached a minimum of 0.6 nM at pH 7.5 in the presence of excess CaCl₂. In cells suspended in sucrose, the affinity of the binding protein for folate increased 20-fold by decreasing the pH from 7.5 to 4.5, indicating that protons can partially fulfill the cation requirement. These results suggest that the folate transport protein of L. casei may contain both a substrate- and cation-binding site and that folate binds with a high affinity only after the cation-binding site has been occupied. The presence of these binding sites would support the hypothesis that folate is transported across the cell membrane via a cation-folate symport mechanism.     

Studies of the blood chemistry of Electrophorus electricus and a new physiological saline solution based thereon

Based upon analyses of the composition of electric eel blood serum we suggest a new physiological saline solution as follows: 188 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 0.15 mM NaH₂PO₄, 1.45 mM Na₂HPO₄ and 5 mM glucose; pH 7.4. The major difference between this saline and that used in most of the previous investigations using eel electroplaques is that the total Na⁺ concentration is increased from between 162.7 and 171.7 mequiv/l to 191 mequiv./l. This increase does not appear to affect the electrophysiological properties of the electroplaque.     

An improved method of transformation in Pseudomonads

Summary A protocol for producing competent Pseuclomonas aeruginosa, Pseudomonas putida, and Xanthomonas maltophilia was adapted and modified from existing methods. Cells were incubated on ice for 30 minutes in buffered 100 mM MgCl₂ followed by 30 minutes in buffered 100 mM CaCl₂ prior to addition of DNA. The MgCl₂-CaCl₂ incubation increased transformation efficiency two to three times, compared with protocols which use incubation in either Mg²⁺ or Ca²⁺, but not both.     

Effects of Cations and PH on Antimicrobial Activity of Thanatin and s-Thanatin Against Escherichia coli ATCC25922 and B. subtilis ATCC 21332

This study analyzes the in vitro effects of cations and pH on antimicrobial activity of thanatin and s-thanatin against Escherichia coli ATCC25922 and B. subtilis ATCC21332. Thanatin and s-thanatin were synthesized by the solid-phase method using a model 432A synthesizer. The bacterial strains tested included two antibiotic-susceptible strains of Escherichia coli ATCC25922 and B. subtilis ATCC21332. Susceptibility determinations were carried out either in a variety of cation concentrations or in pH conditions from pH 5 to pH 8. NaCl or KCl was added to the media to final concentrations of 0, 10, 50, 100, 200, and 500 mM, whereas CaCl₂ and MgCl₂ were added to the media to final concentrations of 0, 1, 2, 5, 10, and 20 mM. The antimicrobial activity of thanatin and s-thanatin against Escherichia coli ATCC25922 and B. subtilis ATCC21332 decreased, as indicated by the increasing minimal inhibitory concentrations (MICs) of both peptides with increasing concentrations of Na⁺/K⁺/Ca²⁺/Mg²⁺. Both peptides lost their activities at 500 mM Na⁺/K⁺ but retained them at 20 mM Ca²⁺/Mg²⁺. Both peptides have MICs that are not significantly different at a variety of pH levels, with the antimicrobial activity slightly higher in neutral or slightly basic media than under acidic conditions. The antimicrobial peptides thanatin and s-thanatin, which have an anti-parallel β-sheet constrained by disulfide bonds, were salt sensitive against both Gram-positive and Gram-negative pathogens in vitro. Determining the reason why the thanatins are salt sensitive would be useful to provide an understanding of how thanatin and s-thanatin kill bacteria. Futher investigation of the antimicrobial properties of these peptides is warranted. G. Wu and J. Ding contributed equally to this article.     

Divalent cations and chlorpromazine can induce non-bilayer structures in phosphatidic acid-containing model membranes

(1) The structural organization of aqueous dispersions of 1,2-dioleoylphosphatidic acid has been investigated by freeze-fracture electron microscopy in relation to variations in pH, divalent cations and the local anaesthetic chlorpromazine. (2) In the pH range 4–8 in the presence of 100 mM Na⁺, dioleoylphosphatidic acid is organized in bilayers. (3) At pH 6 and not at pH 4 and 8.5 addition of Ca²⁺, Mg²⁺, Mn²⁺ and chlorpromazine results in the formation of the hexagonal H_II phase. (4) Ca²⁺ and chlorpromazine addition to mixed phosphatidylcholine-dioleoylphosphatidic acid bilayers at pH 6 results in the formation of lipidic particles.