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PubMed1995

A monovalent cationic conductance that is blocked by extracellular divalent cations in Xenopus oocytes.

1. Native Xenopus oocytes were voltage clamped and exposed to Ringer solutions containing low concentrations of divalent cations. Oocytes, held at -60 mV, developed a reversible non-inactivating smooth inward current (Ic) associated with an increase in membrane conductance. 2. Ic was selectively carried by cations (Na+, K+), indicating that the current was not the result of a non-specific membrane breakdown, but was due instead to removal of a blocking effect of divalent cations on a specific population of endogenous ionic channels located in the oocyte membrane. 3. The blocking effects of Ca2+ and Mg2+ were voltage dependent, implying action at a binding site within the pore of the cationic channel. For example, the half-maximal inhibition (IC50) of Ic by Ca2+ was 61 microM in oocytes held at -60 mV and 212 microM in oocytes held at 0 mV. 4. The Ic channels could be unblocked by depolarization of the membrane even in the presence of physiological concentrations of Ca2+ or Mg2+. The unblocking of the channels was observed as a slowly developing outward current. 5. The novel cationic current was substantially reduced following in vitro maturation of oocytes by treatment with progesterone (10 microM, 4-5 h). 6. The physiological role of Ic channels remains to be elucidated. Nonetheless, their characteristics explain the ionic basis of the sensitivity of oocytes to reductions in extracellular divalent cations and raise the possibility that the channels play a role in calcium homeostasis.

Animals↗
PubMed1979

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.

Bacteria↗
PubMed1979

Calculation of the concentrations of free cations and cation-ligand complexes in solutions containing multiple divalent cations and ligands.

The method described permits the computation of the concentrations of free ions and ion-ligand complexes in a solution containing arbitrary numbers of divalent cations and ligands. It is required that the pH be known, along with appropriate sets of ligand-hydrogen and ligand-divalent cation concentration binding constants. It is assumed that these sets of constants are chosen to be consistent with the ionic strength of the complete solution which contains the divalent cations and ligands. The technique is an iterative one which provides upper and lower bounds for the values of the unknowns. The method does not require initial guesses at the values of the unknowns, and it gives correct answers even when the concentrations involved are many orders of magnitude apart. The present formulation of the problem is restricted to the case where only one cation can bind to a given ligand at any one time. The method is applicable to large molecules with multiple "sub-ligands" provided these sub-ligands are independent in their function as ion-binding sites. These sub-ligands need not all have the same properties. It is also shown that a simple modification of the method permits the determination of the subset of total ion concentrations that are required in order to produce a specified subset of free ion concentrations. The modifications required to include monovalent cation binding are presented in outline form.

Adenosine Triphosphate↗
PubMed1992

Divalent cation block and competition between divalent and monovalent cations in the large-conductance K+ channel from Chara australis.

The patch-clamp technique is used to investigate divalent ion block of the large-conductance K+ channel from Chara australis. Block by Ba2+, Ca2+, Mg2+, and Pt(NH3)4(2+) from the vacuolar and cytoplasmic sides is used to probe the structure of, and ion interactions within, the pore. Five divalent ion binding sites are detected. Vacuolar Ca2+ reduces channel conductance by binding to a site located 7% along the membrane potential difference (site 1, delta = 0.07; from the vacuolar side); it also causes channel closures with mean a duration of approximately 0.1-1 ms by binding at a deeper site (site 2, delta = 0.3). Ca2+ can exit from site 2 into both the vacuolar and cytoplasmic solutions. Cytoplasmic Ca2+ reduces conductance by binding at two sites (site 3, delta = -0.21; site 4, delta = -0.6; from the cytoplasmic side) and causes closures with a mean duration of 10-100 ms by binding to site 5 (delta = -0.7). The deep sites exhibit stronger ion specificity than the superficial sites. Cytoplasmic Ca2+ binds sequentially to sites 3-5 and Ca2+ at site 5 can be locked into the pore by a second Ca2+ at site 3 or 4. Ca2+ block is alleviated by increasing [K+] on the same side of the channel. Further, Ca2+ occupancy of the deep sites (2, 4, and 5) is reduced by K+, Rb+, NH4+, and Na+ on the opposite side of the pore. Their relative efficacy correlates with their relative permeability in the channel. While some Ca2+ and K+ sites compete for ions, Ca2+ and K+ can simultaneously occupy the channel. Ca2+ binding at site 1 only partially blocks channel conduction. The results suggest the presence of four K+ binding sites on the channel protein. One cytoplasmic facing site has an equilibrium affinity of 10 mM (site 6, delta = -0.3) and one vacuolar site (site 7, delta less than 0.2) has low affinity (greater than 500 mM). Divalent ion block of the Chara channel shows many similarities to that of the maxi-K channel from rat skeletal muscle.

Barium↗
PubMed2006

Condensation of nonstochiometric DNA/polycation complexes by divalent cations.

This study found that divalent cations induced the further condensation of partially condensed DNA within nonstochiometric polycation complexes. The addition of a few mmol of a divalent cation such as calcium reduced by half the inflection point at which DNA became fully condensed by poly-L-lysine (PLL) and a variety of other polycations. The effect on DNA condensation was initially observed using a new method, which is based on the concentration-dependent self-quenching of fluorescent moieties (e.g., rhodamine) covalently linked to the DNA backbone at relatively high densities. Additional analyses, which employed ultracentrifugation, dynamic light scattering, agarose gel electrophoresis, and atomic force microscopy, confirmed the effect of divalent cations. These results provide an additional accounting of the process by which divalent cations induce greater chromatin compaction that is based on the representation of chromatin fibers as a nonstoichiometric polyelectrolyte complex. They also offer a new approach to assemble nonviral vectors for gene therapy.

Calcium↗
PubMed1987

Production of iota toxin by Clostridium spiroforme: a requirement for divalent cations.

The effects of divalent cations (Ca2+, Co2+ and Zn2+) on the production of iota toxin by Clostridium spiroforme were studied. Toxin production had an absolute requirement for one or more cations in the range 1-5 mM. Using bispecific antisera, we showed that production of both the components of the toxin (ia and ib) were enhanced by divalent cations added to brain-heart infusion supplemented with peptone and glucose.

ADP Ribose Transferases↗
PubMed1987

A major difference between serum and fibronectin in the divalent cation requirement for adhesion and spreading of BHK21 cells.

Adhesion and spreading of BHK21 cells on adsorbed bovine and foetal bovine serum require addition to the medium of a divalent cation. Divalent cations are effective in the order Mn2+ greater than Co2+ greater than Mg2+ greater than Ca2+, with Ca2+ ineffective below 10(-4)M. On purified fibronectin, however, no added divalent cation is required, since the requirement is largely met by adventitious Ca2+ (circa 10(-5)M) in nominally divalent cation-free saline. In such background Ca2+, adhesion and spreading on fibronectin are only slightly slower than in optimal Mg2+, and appear identical, morphologically, and in sensitivity to cytochalasin D. Cells also spread on fibronectin in response to Mg2+, when Ca2+ is buffered below 10(-6)M, showing that external Ca2+ is not needed as a source for an increase in internal Ca2+. Cells can be induced to spread on serum in low Ca2+ by substantially increasing the fibronectin concentration; this supports other evidence that at its unsupplemented concentration, fibronectin contributes little to the spreading of these cells on serum. The Ca2+ requirement for spreading on preparations of vitronectin (serum spreading factor), partially purified from bovine serum, is similar to that on whole serum. Thus the difference in divalent cation requirement between serum and fibronectin may arise because, on serum, the dominant protein responsible for induction of spreading is vitronectin rather than fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Evidence for four capital and six auxiliary cation-binding sites on calmodulin: divalent cation interactions monitored by direct binding and microcalorimetry.

Recently, Mills and Johnson [7] and our group [9] provided evidence that calmodulin contains, in addition to the four Ca2+-binding sites (capital sites), which are essential for drug- and enzyme-binding, a number of divalent cation-binding sites of different ion selectivity (auxiliary sites), which modulate drug-binding as well as the affinity of Ca2+ for the capital sites. In the present study, the number of auxiliary sites and their relationship to the capital sites were determined by equilibrium gel filtration and by flow microcalorimetry with Zn2+ and Mn2+ as selective probes for the auxiliary sites and with Cd2+ as a probe for both types of sites. In the absence of other divalent cations, 6 mol of Zn2+ bind to calmodulin with an identical affinity constant of 2,850 M-1 and a delta H0 of 106 kJ/mol calmodulin. In the presence of millimolar free Ca2+ calmodulin binds, in addition to four Ca2+, six Zn2+ with an affinity constant of 1,200 M-1 and a delta H0 of 47 kJ/mol calmodulin. The Zn2+-Ca2+ antagonism is governed by negative free energy coupling between the capital and auxiliary sites. In contrast, the Zn2+-Mg2+ antagonism follows the rule of straight competition at all six auxiliary sites. Mn2+ also binds exclusively to the auxiliary sites with affinity constants of 800 or 280 M-1 and delta H0 of 45 or 46 kJ/mol calmodulin in the absence and presence of saturating [Ca2+], respectively. Cd2+ binds to the capital sites with an affinity constant of 3.4 10(4) M-1 (delta H = 35 kJ/mol calmodulin) and to the auxiliary sites with ca. 100-fold lower affinity. The Zn2+ much greater than Mn2+ greater than or equal to Cd2+ greater than Mg2+ selectivity of the auxiliary sites corroborates the potencies of these cations in modulating drug binding. The auxiliary site-specific cations are unable to promote high-affinity complex formation between calmodulin and melittin.

Animals↗
PubMed1984

Generation of an unusual depolarizing response in rabbit primary afferent neurones in the absence of divalent cations.

The effects of divalent cations on responses to 5-hydroxytryptamine (5-HT), gamma-aminobutyric acid (GABA) and 1,1-dimethyl-4-phenyl piperazinium (DMPP) were investigated using a sucrose-gap method to record population responses. In Ca-free medium responses to 5-HT were enhanced, those to DMPP depressed and those to GABA unchanged. In Mg-free medium responses to 5-HT were unchanged, while those to DMPP and GABA were depressed. Removal of both Ca and Mg from the superfusion medium caused a small reduction of GABA responses and a large reduction of DMPP responses. Responses to 5-HT were not only greatly potentiated but were changed in character; the depolarizing phase became sigmoid and the dose dependence between quantity of 5-HT and response magnitude was lost as if 5-HT were triggering an all-or-nothing phenomenon. Dose--response relationships for GABA were normal in the large majority of preparations. In about 10% of preparations, supramaximal amounts of GABA or DMPP evoked large responses of a similar character to those evoked by 5-HT. The large responses, generated by an unknown mechanism, were termed X responses. Further reduction in tissue divalent cations by EGTA (1 mM) caused X responses to be generated spontaneously. Ca, Mg, Mn or Co (1 mM) could suppress X responses. DMPP responses, reduced in Ca/Mg-free medium, were largely restored by 1 mM-Ca. Depression of GABA responses in Ca/Mg-free medium could be entirely attributed to the absence of Mg, Mn being able to substitute for Mg. X responses were generated only after equilibration for 1 h with Ca/Mg-free medium. Attempts to manipulate [Ca]i with dinitrophenol or caffeine did not produce the conditions under which X responses were generated. Intracellular records of responses to 5-HT, GABA or DMPP showed that cells with A fibres responded to GABA but not to 5-HT or DMPP. Fifty-four out of sixty-seven cells with C fibre axons (80%) were depolarized by 5-HT, thirty-seven out of forty-nine (76%) by DMPP and forty out of fifty-seven (70%) by GABA. Eighteen out of thirty-eight (47%) C cells were depolarized by all three agents. Some C cells were very sensitive to 5-HT, 10(-6) M evoking a substantial response. In most, responses to 10(-5) M-5-HT had a slower rate of rise than responses to 10(-4) or 10(-3) M-GABA or DMPP, yet lower 5-HT concentrations normally elicited X responses in sucrose-gap experiments whereas GABA or DMPP normally did not.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1996

Spectroscopic properties of fluorescence dye fura-2 with various divalent cations.

We examined the effects of divalent cations on the fluorescence excitation spectra of fura-2 from a 2 ml solution containing 1 microM fura-2 with an excitation wavelength of 300-425 nm and an emission wavelength of 500 nm. The fluorescence spectra in solutions of fura-2-Ca2+, fura-2-Ba2+, and fura-2-Sr2+ complexes show almost the same pattern. The dissociation constants (Kd) of Ba2+ and Sr2+ with fura-2 were 1.63 and 9.02 microM, respectively, and these values were higher than that of the Ca2+ complex (222 nM). The effect of Mg2+ binding on the fluorescence of fura-2 was quite small, and Mn2+ quenched the fluorescence of fura-2. The spectra of the fura-2-Ca2+ complex were changed by the co-existence of another divalent cation. Divalent cations competitively bind to the binding site of fura-2 and show the respective fluorescence spectra; consequently, the composed fluorescence spectra according to the respective Kd.

Cations↗
PubMed1987

The superstructure of chromatin and its condensation mechanism. III: Effect of monovalent and divalent cations X-ray solution scattering and hydrodynamic studies.

Solutions of rat liver and chicken erythrocyte chromatin at different ionic strengths were characterized by synchrotron X-ray solution scattering, ultracentrifugation, density and viscosity measurements. Previous observations on nuclei were extended to rat liver, calf thymus and yeast nuclei. It is shown that with monovalent cations condensation is independent of the nature of the cation whereas with divalent cations there are significant differences related to the preference of base binding over phosphate binding. The consistency of hydrodynamic and scattering results confirm the view that chromatin in solution at low ionic strength has a helix-like superstructure. A survey of X-ray and neutron scattering results in the literature shows that previous interpretations, e.g. in terms of a 10 nm filament, are incompatible with the experimental data at low resolution.

Animals↗
PubMed1988

The 'cation-dependent' mannose 6-phosphate receptor binds ligands in the absence of divalent cations.

The requirement of divalent cations for binding of the 46 kDa mannose 6-phosphate receptors to phosphomannan and pentamannose 6-phosphate-substituted bovine serum albumin was examined. Receptors from human liver and human brain bound to both affinity ligands in the absence or presence of divalent cations with similar efficiency. The requirement for divalent cations therefore appears not to be necessary for the binding.

Brain↗
PubMed2002

An antiport mechanism for a member of the cation diffusion facilitator family: divalent cations efflux in exchange for K+ and H+.

Members of the cation diffusion facilitator (CDF) family of membrane transport proteins are found in eukaryotes and prokaryotes. The family encompasses transporters of zinc ions, with cobalt, cadmium and lead ions being additional substrates for some prokaryotic examples. No transport mechanism has previously been established for any CDF protein. It is shown here that the CzcD protein of Bacillus subtilis, a CDF protein, uses an antiporter mechanism, catalysing active efflux of Zn2+ in exchange for K+ and H+. The exchange is probably electroneutral, energized by the transmembrane pH gradient and oppositely oriented gradients of the other cation substrates. The data suggest that Co2+ and Cd2+ are additional cytoplasmic substrates for CzcD. A second product of the same operon that encodes czcD has sequence similarity to oxidoreductases and is here designated CzcO. CzcO modestly enhances the activity of CzcD but is not predicted to be an integral membrane protein and has no antiport activity of its own.

Antiporters↗
PubMed1994

Mammalian urinary bladder permeability is altered by cationic proteins: modulation by divalent cations.

It was previously demonstrated that protamine sulfate (PS, a cationic polypeptide) as well as synthetic cationic polypeptides (CpP, e.g., polylysine and polyarginine) caused an increase in the apical membrane conductance of the mammalian urinary bladder epithelium that was voltage dependent. The membrane conductance induced by these CpP was mediated by a saturable binding site and was partially blocked by CpP (self-inhibition). The PS-induced membrane conductance can be modified by polyvalent cations at three sites. The first site was to competitively inhibit the interaction of PS with an apical membrane binding site. The second site was to reversibly block the conductance induced by PS. The relative binding affinity (block of PS-induced conductance) sequence was as follows: UO2(2+) > La3+ > Mn2+ > Ba2+ > or = Ca2+ > Sr2+. Although La3+, Mn2+, Ba2+, Ca2+, and Sr2+ inhibited > or = 81% of the PS-induced conductance, UO2(2+) inhibited only 51% and Mg2+ was without effect. The third site was to increase the rate of loss of the PS-induced conductance from the apical membrane. Although neither carbodiimides (carboxyl group reactive reagents) nor neuraminidase (cleaves sialic acid residues) altered the effect of PS on the urinary bladder conductance, PS increased the conductance of lipid bilayers composed of negatively charged phospholipids. A candidate for the binding site might be the negatively charged phosphate groups of membrane lipids.

Animals↗
PubMed1992

Ionic modulation of the effects of heparin on plasminogen activation by tissue plasminogen activator: the effects of ionic strength, divalent cations, and chloride.

Ionic strength, divalent cations, and Cl- modulate the ability of the glycosaminoglycan heparin to stimulate the activation of human plasminogen (Pg) by tissue-type Pg activator. Kinetic analysis of Pg activation indicates that heparin is inhibitory, stimulatory, or nonstimulatory as a function of ionic strength. While increasing ionic strength inhibits Pg activation in the absence of heparin, in it presence an activation phase followed by an inhibitory phase is observed. Divalent cations, inhibitors of activation in the absence of heparin, increase the rate of activation in its presence. Kinetic analysis demonstrates that divalent cations augment the heparin stimulatory effect a maximum of 60-fold due to increases in kcat without changes in Km of the reaction. This effect is heparin-specific, since activation is not affected by Ca2+ in the presence of heparan sulfate or de-N-sulfated heparin. Also, Cl- inhibits Pg activation in the presence of heparin by acting as a competitive inhibitor (Kic of 100 mM). Furthermore, inhibition by Cl- reduces the overall magnitude of heparin stimulation of Pg activation. These results suggest that physiologic ions in combination with heparin may be significant effectors of Pg activation in the vascular microenvironment.

Binding, Competitive↗
PubMed1995

Do pancreatic islet cells from neonatal rats have surface receptors or sensors for divalent cations?

The effects of extracellular divalent cations on the intracellular Ca2+ concentration ([Ca2+]i) in neonatal rat islet cells were investigated to determine whether these cells, like several others, have signal-generating surface cation sensors. Raising the external Ca2+ concentration by 1 mM increments triggered either sustained increases in [Ca2+]i or large sharp [Ca2+]i spikes followed by return to a suprabasal level. The external Ca(2+)-triggered [Ca2+]i responses were abolished by treating the cells with the inhibitor of inositol phospholipid hydrolysis, neomycin (1.5 mM), but not by another phospholipase C inhibitor, U-73,122 (2.5 microM), or the voltage-sensitive Ca2+ channel blockers nifedipine (20 microM) and methoxyverapamil (D600; 50 microM). [Ca2+]i responses were also triggered by barium (Ba2+; 1 mM) and cobalt (Co2+; 1 mM). The Ba2+ responses were also inhibited by neomycin and unaffected by nifedipine or D600 and the Co2+ response required external Ca2+. Therefore, neonatal rat pancreatic islet cells may display divalent cation receptors/sensors on their surfaces. Activation of these putative receptors, which are coupled to neomycin-sensitive, voltage-independent, dihydropyridine-insensitive channels, by Ca2+, Ba2+ or Co2+ would trigger [Ca2+]i responses by opening these channels to admit external Ca2+ into the cell. The physiological function(s) of such cell-surface divalent cation receptors/sensors and the [Ca2+]i surges they generate in pancreatic islet cells is not known.

Animals↗
PubMed1984

Modulation of myosin filament conformation by physiological levels of divalent cation.

The effect of divalent cation, in particular Mg2+, on the properties of synthetic myosin filaments has been studied; and substantial changes in sedimentation and light scattering demonstrated to occur in the physiological range of free Mg2+. A pre-requisite for these studies has been the definition of a modified method for the preparation of myosin in highly monodisperse filament form, rigorously free from thin filament proteins. The sedimentation coefficient at infinite dilution shows a large increase (169 S to 193 S) in the range 0.2 mM to 3 mM in Mg2+. The anomalous frictional increment found for these filaments is thus substantially reduced. The concentration dependence (ks), however, shows a substantial decrease (470 ml/g to 334 ml/g) in the same range of Mg2+, and the calculated filament molecular weight is virtually unchanged. A change in the filament conformation is thus indicated. This is confirmed by an analysis of the turbidity of the filaments in the centrifuge cell, which shows a similar increase in response to the addition of Mg2+. These effects have been found to be independent of ionic strength (0.07 to 0.11), pH (7.0 to 7.6), the presence of MgATP or the presence of low levels of Ca2+ (approximately 100 microM). These effects studied indicate the action of Mg2+ through a low-affinity binding site (Kd approximately 1.5 X 10(-3) M). We consider that a significant change in crossbridge conformation can adequately explain these changes in physical and enzymic properties. A provisional model is proposed, in which the effect of Mg2+ is to bring the crossbridges into closer proximity to the filament shaft.

Animals↗
PubMed1982

Circular dichroism (CD) studies on yeast enolase: activation by divalent cations.

The effect of divalent cations on the near ultraviolet circular dichroism (CD) spectrum of yeast enolase showed that calcium, magnesium, and nickel ions produced identical changes. This was interpreted as indicating that the cations bound to the same sites on the enzyme and produced identical changes in tertiary structure. There was no effect of magnesium ion on the far ultraviolet spectrum. Evidently magnesium ion has no effect on the secondary structure. Substrate bound to the enzyme when the above cations were present although calcium permits no enzymatic activity. The CD spectral difference produced by the substrate was nearly the reverse of that produced by the metal ions. Glycolic acid phosphate, a competitive inhibitor lacking carbon-3, produced no effect, indicating carbon-3 was necessary for the CD spectral changes. The CD and visible absorption spectra of nickel and cobalt bound to various sites on the enzyme showed that the binding sites were octahedral or distorted octahedral in coordination and that the ligands appeared to be oxyligands: water molecules, hydroxyl or carboxyl groups. Examination of the effects of substrate and two compounds thought to be "transition state analogues" showed that these perturbed the "conformational" sites of the enzyme. The "catalytic" and "inhibitory" sites did not appear to be very CD active.

Cations, Divalent↗