Effects of divalent cations on phosphatase secretion in cultured tobacco cells

Some differences were found between Mg²⁺- and Ca²⁺-stimulated phosphatase secretion in cultured tobacco cells. The effect of Mg²⁺ ions was greater than that of Ca²⁺ ions, and Ca²⁺ ions at below 1 mM rather depressed the secretion. Upon the addition of Mg²⁺ ions plus Ca²⁺ ions, a synergistic stimulation of the secretion occurred. Different influences on the effects of Mg²⁺ and Ca²⁺ ions on the secretion were exerted by treating cells with metabolic inhibitors that reduced the level of cellular metabolic energy. Phosphate (Pi) and arsenate did not depress the secretion in the presence of Mg²⁺ ions, but did depress it in the presence of Ca²⁺ ions. These results strongly suggested that the secretion of phosphatase involved at least two different steps affected by divalent cations.     

Effects of divalent cations and polyethylene glycol on the membrane fluidity of protoplast

Calcium is often used to stabilize membranes and enhance membrane fusion. We have used the fatty acid spin label, 5-nitroxy stearic acid to measure fluidity changes in the plasma membrane of carrot suspension culture cell protoplasts in response to divalent cations. Electron spin resonance spectra from spin-labeled protoplasts showed no membrane fluidity changes (as determined by the hyperfine splitting constant, 2A_max) in the presence of Mg from 0 to 10 millimolar or Ca from 0 to 5 millimolar. Protoplasts in 10 millimolar Ca, however, showed a dramatic increase of 5 gauss in 2A_max and evidence of exchange-broadening. The original (control) spectrum was regained by removing bound Ca with a Ca chelator. Polyethylene glycol, which enhances protoplast fusion, did not alter the membrane fluidity in the region of the 5-nitroxy stearic acid probe if added simultaneously with or following 10 millimolar Ca. Pretreatment with polyethylene glycol did, however, inhibit the Ca-induced phase separation. These data on a living system describe membrane structural changes under conditions similar to those used for protoplast fusion.     

Effects of divalent cations on toad end-plate channels

Summary Miniature end-plate currents (MEPCs) and acetylcholine-induced current fluctuations were recorded in voltageclamped, glycerol-treated toad sartorius muscle fibers in control solution and in solutions with added divalent cations. In isosmotic solutions containing 20mm Ca or Mg, MEPCs had time constants of decay ( _D ) which were about 30% slower than normal. In isotonic Ca solutions (Na-free), greater increases in both _D and channel lifetime were seen; the null potential was –34 mV, and single-channel conductance decreased to approximately 5 pS. Zn or Ni, at concentrations of 0.1–5mm, were much more effective in increasing _D than Ca or Mg, although they did not greatly affect channel conductance. The normal temperature and voltage sensitivity of was not significantly altered by any of the added divalent cations. Surface potential shifts arising from screening of membrane fixed charge by divalent cations cannot entirely explain the observed increases in , especially when taken together with changes in channel conductance.     

Stabilization effect of protons and divalent cations on membrane structures of lipids

Ordered-fluid phase transitions of methylphosphatidic and phosphatidic acid bilayers are discussed theoretically to explain comprehensively various observed data in the presence of Ca2+ or protons. It is shown that the observed data can be explained reasonably by taking account of the interaction among neighboring head-groups with the aid of Ca2+ or protons. As a result, a quantitative explanation is given for the dependence of the phase-transition temperature on pH as well as Ca2+ concentration with the coexistence of monovalent cations. The dependence of the cooperativity of the transition on pH and monovalent cation concentration is also well explained. It is also pointed out that in the ordered-fluid phase transitions, a hysteresis metastable state and phase separation can be expected to appear under some conditions.     

Effects of divalent cations on thermophilic inorganic pyrophosphatase.

Divalent cations were shown to affect the structure and thermostability of thermophilic inorganic pyrophosphatase [pyrophosphate phosphohydrolase EC 3.6.1.1] purified from Bacillus stearothermophilus and thermophilic bacterium PS-3. The properties of the enzymes from the two sources were found to be very similar. The enzymes were very unstable to heart in the absence of divalent cations, being inactivated gradually even at 40 degrees C. However, they became stable to heat denaturation in the presence of Mg2+, between pH 7.8 and 9.0. Similar induced thermostability was detected when Mn2+, Co2+, Ca2+, Cd2+, and ZN2+ were added, though the latter three cations were not essential for enzyme activity. On adding divalent cations, the optical properties such as absorption spectra, fluorescence spectra, and circular dichroism (CD) were changed. Gel filtration and disc electrophoresis revealed that the molecular weight of both enzymes was 5.4 x 10(4) in Tris-SO4 buffer and 11 x 10(4) in Tris-HCL buffer, suggesting monomer-dimer transformation. In the presence of divalent cations in Tris-SO4 fuffer, the enzymes dimerized; this was confirmed by sedimentation velocity measurements. The enzymes in Tris-HCL buffer did not show thermostability unless divalent cations were added. The results in the present study indicate that binding of divalent cations to each enzyme caused some conformational change in the vicinity of aromatic amino acid residues leading to dimerization of the enzyme molecule so that it became thermostable. It was also suggested that histidyl residues play an important role in the thermostability induced by divalent cations on the basis of the pH dependencies of thermostability and CD spectra.     

Effects of some divalent cations on motoneurones in cats

In cats under Dial, Co, Mn, La, and Sr were injected extracellularly near lumbosacral motoneurones. All tended to improve intracellular recording, but when the membrane potential was initially stable, Mn, and especially Co, had a moderate and reproducible depolarizing action. Both Mn and Co depressed excitatory postsynaptic potentials evoked by dorsal root stimulation. The prominent after-hyperpolarization (a.h.p.), which normally follows the motoneuronal action potential, was consistently and reversibly depressed by Mn and Co (as well as La), the underlying conductance increase being also diminished, but there was no significant reduction in the after-depolarization. By contrast, Sr tended to potentiate the a.h.p., especially when this was depressed by a previous injection of Co or Mn. Unlike the other cations, Co had a marked depressant effect on the action potential, particularly its rate of rise. Since the action potential could be immediately restored by hyperpolarization or by an injection of Sr (in the absence of depolarization), Co may enhance Na inactivation.     

Stimulation and inhibition of secretion in Paramecium: role of divalent cations

The effects of Ca2+ and Mg2+ on exocytosis in Paramecium tetraurelia cells were examined with light microscopy, freeze fracture (FEM) and transmission electron microscopy (TEM) of thin-sectioned embedded cells. Picric acid-Ca2+-induced secretion in wild type (wt) cells was captured by "quick" fixation with OsO4, and TEM demonstrated membrane fusion occurring before trichocyst matrix (tmx) expansion. Cells stimulated with picric acid in the presence of high extracellular Mg2+ showed very few sites of membrane fusion and no tmx expansion, suggesting that Ca2+ is required for both membrane fusion and tmx expansion. Further information was obtained by comparing secretory responses of wt cells with a temperature-sensitive secretory mutant, nd 9. These cells when grown at the permissive temperature (18 degrees C) possess normal rosettes at the secretory site and secrete in response to picric acid-Ca2+, but when grown at 27 degrees C they lack rosettes and do not secrete (Beisson, J., M. Lefort-Tran, M. Pouphile, M. Rossignol, and B. Satir, 1976, J. Cell Biol., 69:126-143). Quantitation of picric acid-Ca2+-induced secretion revealed that: (a) the number of tmx secreted by wt and nd 9 cells was independent of their cultural growth phase, (b) wt cells secreted the same number of tmx when grown either at 18 or 27 degrees C, and (c) nd 9 18 degrees C cells secreted the same number of tmx as wt 18 or 27 degrees C cells. Wild type and nd 9 cells had the same frequencies of occupied and unoccupied secretory sites as determined by quantitative analysis of freeze-fracture replicas. After stimulation with divalent cation ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and Ca2+, wt cells showed a significant reduction in the frequency of occupied sites. FEM and TEM studies revealed that {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187-Ca2+ induced tmx expansion and normal fusion of the plasma and trichocyst membranes in wt and nd 9 18 degrees C cells, but induced tmx expansion without concomitant membrane fusion in nd 9 27 degrees C cells. The lack of membrane fusion in nd 9 27 degrees C cells suggests that the molecules represented by rosette particles are required specifically for membrane fusion.     

Control of membrane permeability in animal cells by divalent cations

The permeability of several cell lines, including HeLa, L929, 3T6 and 3T3, to various compounds is affected by the concentration of divalent cations in the culture medium. In the absence of Mg2+ ions but with 4-8 mM CaCl2 in the medium, HeLa and L929 cells become permeabilized, as measured by the entry of the aminoglycoside antibiotic hygromycin B. However, 3T3 and 3T6 cells become much more permeable when calcium and magnesium are both absent from the medium. Addition of Mg2+ above 2 mM abolishes the permeabilization induced by Ca2+. Basic pH favors permeabilization, whereas acidic pH inhibits the entry of hygromycin B. Increased entry of macromolecules, such as the toxin alpha-sarcin, horseradish peroxidase (HRP) and luciferase, is also observed under permeabilization conditions, suggesting that this method could be of general use, since it is not harmful to cells and is fully reversible. Exit of 86Rb+ ions and [3H]uridine-labelled nucleotides was also assayed. We did not observe increased release of these compounds from preloaded cells under various calcium concentrations. Finally, the effects of several inhibitors of endocytosis and other membrane functions on the permeabilization inhibitors of endocytosis and other membrane functions on the permeabilization process were also analysed. The entry of alpha-sarcin was not affected by nifedipine, dibucaine or mepacrine, but was partially inhibited by NH4Cl, amantadine and chloroquine.     

Effect of histamine and divalent cations on the activity and stability of tryptase from human mast cells

Tryptase from human mast cells is stabilized by negatively charged macromolecules such as heparin and is not affected by the protein inhibitors of serine proteinases normally present in human extracellular fluids. The current study demonstrated inhibition of tryptase-catalyzed cleavage of tosyl-Gly-Pro-Lys-p-nitroanilide by histamine and calcium, and destablization only by calcium. Calcium-mediated inhibition was competitive with a Ki of 30 mM. Cooperation of calcium with other extracellular cations or concentrations of calcium possible within cells or granules may permit calcium-mediated inhibition to occur in vivo. In contrast, only 5 mM calcium is needed to cause an irreversible 50% loss of tryptase activity after 60 min at room temperature. Histamine and N-methyl histamine concentrations of 2 mM to 10 mM inhibited tryptase activity by a different mechanism than calcium, resulting in sigmoid rather than hyperbolic kinetics. Whether this reflects cooperative binding of histamine to tryptase or conformational alterations of tryptase is not known. These concentrations of histamine are most relevant to those in mast cell secretory granules estimated at 100 mM, where tryptase is stored fully active and where histamine may play a role in attenuating tryptase activity.     

A neutral carrier-based liquid membrane microelectrode for divalent putrescine cations

A new ion-selective liquid membrane microelectrode, based on the neutral carrier 1,1′-bis(2,3-naphtho-18-crown-6), is described that shows the dependence of EMF on the activity of divalent putrescine cations a _Put, with the linear slope s _Put = 26 ± 3 mV/decade (mean ± SD, N = 18), in the range 10⁻⁴–10⁻¹ M at 25 ± 1 °C. Values of potentiometric putrescine cation selectivity coefficients of logK ^Pot _{Put j} (mean ± SD, N) are obtained by the separate solution method for the ions K⁺ (1.0 ± 0.4, 10), Na⁺ (−1.2 ± 0.4, 8), Ca²⁺ (−2.3 ± 0.5, 10) and Mg²⁺ (−2.5 ± 0.5, 7). The microelectrode can be applied for the direct analysis of the activities of free divalent putrescine cations in the range 5 × 10⁻⁴ to 10⁻¹ M in an extracellular ionic environment. Established analytical methods, e.g. high performance liquid chromatography, determine the total concentration of the derivatives of free and bound putrescine. Received: 20 December 1998 / Revised version: 7 May 1999 / Accepted: 27 May 1999     

Effects of divalent cations on dynein cross bridging and ciliary microtubule sliding

We recently demonstrated that addition of the divalent cation Mg++ to demembranated cilia causes the dynein arms to attach uniformly to the B subfibers. We have now studied the dose-dependent relationship between Mg++ or Ca++ and dynein bridging frequencies and microtubule sliding in cilia isolated from Tetrahymena. Both cations promote efficient dynein bridging. Mg++-induced bridges become saturated at 3 mM while Ca++-induced bridges become saturated at 2 mM. Double reciprocal plots of percent bridging vs. the cation concentration (0.05-10 mM) suggest that bridging occurs in simple equilibrium with the cation concentration. When microtubule sliding (spontaneous disintegration in 40 mM N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES), 0.1 mM ATP at pH 7.4) is assayed (A350 nm) relative to the Mg++ or Ca++ concentration, important differential effects are observed. 100% Disintegration occurs in 0.5-2 mM Mg++ and the addition of 10 mM Mg++ does not inhibit the response. The addition of 0.05-10 mM Ca++ to cilia reactivated with 0.1 mM ATP causes a substantial reduction in disintegration at low Ca++ concentrations and complete inhibition at concentrations greater than 3 mM. When Ca++ is added to cilia reactivated with 2 mM Mg++ and 0.1 mM ATP, the percent disintegration decreases progressively with the increasing Ca++ concentration. The addition of variable concentrations of Co++ to Mg++-activated cilia causes a similar but more effective inhibition of the disintegration response. These observations, when coupled with the relatively high concentrations of Ca++ or Co++ needed to inhibit disintegration, suggest that inhibition results from simple competition for the relevant cation-binding sites and thus may not be physiologically significant. The data do not yet reveal an interpretable relationship between percent disintegration, percent dynein bridging, and percent ATPase activity of both isolated dynein and whole cilia. However, they do illustrate that considerable (sliding) disintegration (60%) can occur under conditions that reveal only 10-15% attached dynein cross bridges.     

Effects of divalent cations on encapsulation and release in the GroEL-assisted folding

Chaperonin GroEL assists protein folding in the presence of ATP and magnesium. Recent studies have shown that several divalent cations other than magnesium induce conformational changes of GroEL, thereby influencing chaperonin-assisted protein folding, but little is known about the detailed mechanism for such actions. Thus, the effects of divalent cations on protein encapsulation by GroEL/ES complexes were investigated. Of the divalent cations, not only magnesium, but also manganese ions enabled the functional refolding and release of 5,10-methylenetetrahydroforate reductase (METF) by GroEL. Neither ATP hydrolysis nor METF refolding was observed in the presence of zinc ion, whereas only ATP hydrolysis was induced by cobalt and nickel ions. SDS-PAGE and gel filtration analyses revealed that cobalt, nickel and zinc ions permit the formation of stable substrate-GroEL-GroES cis-ternary complexes, but prevent the release of METF from GroEL.     

Effects of divalent cations on in vitro maturation of bovine oocytes

Lowering the external concentrations of both Mg+2 and Ca+2 caused failure of meiotic resumption in vitro of bovine, oocyte-cumulus complexes. Lowering of external Ca+2 levels singly had no effect on either meiotic resumption or completion of the first meiotic division. Lowering of external Mg+2 concentrations alone, although having no effect on meiotic resumption in vitro when Ca+2 was present, did interfere with the completion of the first meiotic division. The result was arrest of oocyte maturation between germinal vesicle breakdown and formation of the first metaphase plate.     

Adsorption of divalent cations on DNA

The distribution of divalent ions in semidilute solutions of high-molecular-mass DNA containing both sodium chloride and strontium chloride in near-physiological conditions is studied by small-angle x-ray scattering and by small-angle neutron scattering. Both small-angle neutron scattering and small-angle x-ray scattering reveal a continuous increase in the scattering intensity at low q with increasing divalent ion concentration, while at high q the scattering curves converge. The best fit to the data is found for a configuration in which DNA strands of cross-sectional radius 10 angstroms are surrounded by a counterion sheath of outer radius approximately 13.8 angstroms, independent of the strontium chloride concentration. When the strontium chloride is replaced by calcium chloride, similar results are obtained, but the thickness of the sheath increases when the divalent salt concentration decreases. These results correspond in both cases to partial localization of the counterions within a layer that is thinner than the effective Debye screening length.     

Effects of nucleotides and divalent cations on phospholipase activity in Saccharomyces cerevisiae

Divalent cations activate the lysophospholipase and transacylase reactions catalyzed by the same enzymes in the yeast Saccharomyces cerevisiae. The activation was observed at neutral pH, but not at the pH optimum of lysophospholipase/transacylase, near 3.5. Adenine nucleotides, especially AMP and ADP, are strong inhibitors of the same group of enzymes. Half maximal inhibition by AMP was found at a concentration of about 20 M. The inhibition by nucleotides in low concentrations is enhanced by divalent cations.     

Effects of manganese and other divalent cations on calcium uptake and catecholamine secretion by primary cultures of bovine adrenal medulla cells

Primary cultures of bovine adrenal medullary chromaffin cells were used to examine the effect of replacing divalent cations in the extracellular media on secretion. When calcium was replaced by manganese, nicotine-stimulated secretion was delayed in onset for 3 to 5 minutes, but continued for approximately 60 minutes. In contrast, calcium-supported secretion began immediately on stimulation and plateaued by 10 minutes. ⁵⁴Mn²⁺ uptake occurred on stimulation but at a lower rate than ⁴⁵Ca²⁺ uptake. There was no delay of ⁵⁴Mn²⁺ uptake upon stimulation and ⁵⁴Mn²⁺ uptake was considerably prolonged compared to ⁴⁵Ca²⁺ uptake. Replacement of calcium with strontium gave results similar to those with calcium, and, in addition, strontium was able to bring about secretion by itself in a manner similar to barium. Inhibition experiments showed that the potency for inhibiting calcium uptake was Cd²⁺>Mn²⁺>Ca²⁺>Sr²⁺.