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PubMed2006

DNA condensation and interaction with zwitterionic phospholipids mediated by divalent cations.

Artificial viruses are considered to be a promising tool in gene therapy. To find lipid-DNA complexes with high transfection efficiency but without toxicity is a fundamental aim. Although cationic lipids are frequently toxic for cells, neutral lipids are completely nontoxic. Zwitterionic lipids do not interact with DNA directly; however, the interaction can be mediated by divalent cations. Langmuir monolayers represent a well-defined model system to study the DNA-lipid complexes at the air/water interface (quasi-2D systems). In this work, isotherms, infrared reflection absorption spectroscopy (IRRAS), X-ray reflectivity (XR), grazing incidence X-ray diffraction (GIXD), and Brewster angle microscopy (BAM) measurements are used to study the interaction of calf thymus DNA with DMPE (1,2-dimyristoyl-phosphatidylethanolamine) monolayers mediated by Ca2+ or Mg2+ ions. DNA adsorption is observed only in the presence of divalent cations. At low lateral pressure, the DNA partially penetrates into the lipid monolayer but is squeezed out at high pressure. The adsorption layer has a thickness of 18-19 A. Additionally, GIXD provides information about a one-dimensional ordering of adsorbed DNA. The periodic distance between DNA strands depends on the type of the divalent cation.

Adsorption↗

PubMed1985

Effect of divalent cations on the assembly of neutral and charged phospholipid bilayers in patch-recording pipettes.

Monolayers of the negatively charged phospholipid phosphatidylserine (PS) and of the amphoteric phospholipid dioleoylphosphatidylethanolamine (DOPE) were used to assemble bilayers at the tip of patch-recording pipettes. PS bilayers, with seal resistances in the range of gigaohmns (gigaseals), could only be generated when millimolar concentration of divalent cations, Ca++, Mg++, or Ba++ were present in the pipette and bath solutions. In contrast, gigaseals of DOPE were independent of divalent ion concentration in the pH range where DOPE is predominantly neutral (pH 6.5) or positively charged (pH 1.5). At pH 10.0, when most DOPE molecules bear a net negative charge, gigaseals became divalent cation dependent, in a manner quantitatively similar to that of PS at neutral pH. The results indicate that divalent cations play an important role in stabilizing gigaseals of negatively charged lipid but are of no consequence in neutral or positively charged seals.

Barium↗

PubMed2002

Membrane-bound heparin binding proteins from HL-60 cells purified in a two-step affinity chromatography differentially eluted with divalent cations.

Solubilized membrane proteins from HL-60 cells were separated by two-step affinity chromatography. Proteins eluted with MgCl2 in the first heparin-gel were applied to the second heparin-gel and eluted with CaCl2. The eluted proteins were analysed and purified by electrophoresis. N-terminal amino acid sequences of eight proteins on the characteristic bands were determined. Homology search for the sequences indicated that three microsomal proteins, two nuclear proteins and a glycolytic enzyme were eluted with divalent cations, whereas a nuclear ribonucleoprotein and a membrane-cytoskelton linker protein were not dissociated with divalent cations, but with 2 M NaCl. Heparin affinity chromatography combined with differential elution with divalent cations can be a useful method for separation of membrane proteins.

Amino Acid Sequence↗

PubMed1995

Reaction of 20S proteasome: shift of SDS-dependent activation profile by divalent cations.

The multicatalytic endopeptidase complex (20S proteasome) is a latent high-molecular-mass multisubunit proteinase. In many investigations, SDS has been used as a proteasome activator at some fixed concentration that was apparently optimal. This study examined the effects of various divalent cations on the SDS-dependent peptidase and casein degradation activities of 20S proteasome purified from Xenopus laevis oocytes at a series of SDS concentrations and the correlation between these effects and the critical micelle concentration (CMC) of SDS. Surprisingly, it was found that divalent cations such as Mg2+ markedly shifted the SDS-dependent activation profiles to a lower concentration range. Ca2+, Mn2+, Co2+, and Zn2+ also markedly reduced the optimum SDS concentration in the Suc-Leu-Leu-Val-Tyr-MCA hydrolysis reaction: for example, 5 mM Co2+ reduced the optimum SDS concentration from 0.065 to 0.005%. However, in all cases examined the optimum concentrations were below the CMC. Cu2+, Hg2+, and Cd2+ strongly inhibited the SDS-dependent maximum activity without remarkably shifting the optimum SDS concentration. No correlation between the shift and the inhibition was recognized. Most interestingly, remarkable activation of casein degradation by SDS was observed only by addition of the divalent cations Mg2+, Ca2+, and Mn2+. These cations might be essential for casein degradation. The activation and inactivation ranges of SDS concentration varied with the species of substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1983

Effects of divalent cations and sulfhydryl reagents on the p-aminohippurate (PAH) transporter of renal basal-lateral membranes.

A specific system of transport for p-aminohippurate (PAH) is demonstrated in rabbit renal basal-lateral membrane vesicles. The PAH uptake into an intravesicular space is inhibited by probenecid in concentrations above 0.2 mM. The transport is saturable and is also temperature-dependent with an optimum between 37 and 45 degrees C. Divalent cations are able to enhance the uptake 2- to 3-fold. The stimulatory effect of the divalent cations diminishes in the following order: Mg++ = Mn++, Ba++, Ca++ and Sr++. Maximum stimulation occurs between 2.5 and 5 mM Mg++. The divalent cation stimulatory effect is not the result of changes in the size of the vesicles, in the degree of vesiculation, in the net charge of the membrane or of a transient potential difference across the membrane. Several inhibitors, more inhibitory than probenecid, were found. These are: lithium diiodosalicylate; 4-acetamido-4'-isothiocyano 2,2'-disulfonic acid stilbene; the mercurials, mersalyl acid, p-chloromercuriphenyl sulfonate and Hg++; and 5,5'-dithiobis(nitrobenzoate). Among these, mersalyl acid is the most potent inhibitor for PAH uptake. Its inhibitory effect is probably a combination of its reactivity toward sulfhydryl groups and its anionic character. The results with sulfhydryl reagents indicate that the PAH transport system contains sulfhydryl groups which are essential for the uptake activity. These sulfhydryl groups are probably buried in a hydrophobic region within the lipoprotein matrix of the basal-lateral membrane.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1990

Proton and divalent cations induce synergistic but mechanistically different destabilizations of pH-sensitive liposomes composed of dioleoyl phosphatidylethanolamine and oleic acid.

Protons and divalent cations show synergistic effects on the destabilization of liposomes composed of unsaturated phosphatidylethanolamine and oleic acid (Düzgünes et al., Biochemistry (1985) 24, 3091). We have extended these observations and investigated the effects of Ca2+ and Mg2+ on the proton-induced destabilization of dioleoyl phosphatidylethanolamine/oleic acid (DOPE/OA) (4:1 molar ratio) liposomes. Temperature-induced aggregation was measured by 90 degrees light scattering. Lipid mixing was used to monitor vesicle destabilization and freeze-fracture electron microscopy was used to examine the structures formed from DOPE/OA vesicles in the presence of Ca2+ and/or protons. Both Mg2+ and Ca2+ shift the pH required for 50% lipid mixing to higher values. Temperature-induced vesicle aggregation occurs at lower temperatures in the presence of divalent cations and/or protons, indicating that intervesicular repulsions are decreased. Freeze-fracture electron micrographs show that the structures formed from DOPE/OA in the presence of Ca2+ differ significantly from those found in the presence of protons. In general, protons induce the formation of hexagonal phase, while the presence of Ca2+ leads to the formation of extensive regions of lamellar sheets with numerous lipidic particles. The synergistic effect of divalent cations and proton may be important for the maximal biological activity of DOPE/OA liposomes.

Calcium↗

PubMed1989

Effects of tetrodotoxin and monensin on the noradrenaline output induced by ouabain from the guinea-pig vas deferens in the presence of various divalent cations.

The importance of Na+ in the ouabain-induced increase in noradrenaline output from adrenergic nerve terminals of the guinea-pig vas deferens was investigated during incubation in standard Krebs solution and Sr2+- and Ba2+-substituted Krebs solutions. Ouabain (10(-4) M) caused a gradual increase in noradrenaline output regardless of the type of divalent cation used. The rate of development of the ouabain-induced noradrenaline output and the maximum amount released depended on the divalent cation present (Ba2+ greater than Sr2+ greater than Ca2+). The magnitude of the ouabain-induced response depended on the concentration of extracellular Na+. The sensitivity of the ouabain-induced response to a change in Na+ concentration depended on the divalent cation present (Ca2+ greater than Sr2+ greater than Ba2+). Tetrodotoxin (1.6 x 10(-6) M) delayed the onset, whereas monensin (10(-5) M) hastened it. The maximum level of the ouabain-induced response was attenuated by monensin in all the incubation solutions. These results suggest that Na+ entry into adrenergic nerve terminals through tetrodotoxin-sensitive mechanisms is crucial for the increase in noradrenaline output due to ouabain when extracellular Ca2+ is replaced with Sr2+ or Ba2+.

Animals↗

PubMed1998

Highly selective separation of rhodopsin from bovine rod outer segment membranes using combination of divalent cation and alkyl(thio)glucoside.

The micellization process of bovine rod outer segment (ROS) membranes is investigated utilizing a series of neutral detergents. It is found that when alkyl(thio)glucosides with an appropriate hydrophillic-lipophilic balance (e.g. octylthioglucoside) are used in combination with a divalent cation, rhodopsin is selectively extracted from ROS membranes at a specific detergent-to-membrane ratio. This allows remarkable purification of rhodopsin by a single-step solubilization, because the residual membranes are heavily aggregated in the presence of divalent cation and are therefore easily sedimented by low-speed centrifugation. The absorption spectrum of the supernatant reproducibly exhibits an A280/A500 value of 1.6, an excellent value that could rarely be obtained by chromatographic purification. The degree of purification also depends on the type of divalent cation included in the solubilization solution; specific binding of IIB-series cations (Zn2+ and Cd2+) to ROS membranes is suggested to play an important role in the solubilization process. The present result represents a unique example of selective solubilization of a specific membrane protein from highly aggregated membranes.

Animals↗

PubMed1989

Competitive action of divalent cations and D600 in frog slow muscle fibers.

Single, slow muscle fibers from Rana temporaria were equilibrated in normal Ringer's. 95 mmol/liter K(+)-solution containing various concentrations of Ca2+, Ni2+, Mn2+ or Mg2+ was applied, and the ensuing contractures were recorded isometrically. While peak tension (Fmax) was little affected, maintained tension (measured 1 min after onset of contracture) strongly depended on the concentration and species of divalent cations. Tension was maintained at its peak value in the presence of all species of divalent cations provided their concentrations were adequately increased. Dose-response curves were hyperbolic; Lineweaver-Burk plots revealed straight lines with different slopes intersecting near 1/Fmax, and indicating the following order of efficiency: Ni2+ greater than Ca2+ greater than Mn2+ much greater than Mg2+. Hill plots for these cations resulted in straight lines with slopes near 1. Qualitatively similar relationships were obtained with contracture solutions containing D600 (3-12 mumol/liter). However, under these conditions higher concentrations of Ca2+ or Ni2+ were required in order to fully maintain tension. After a step concentration change in the medium during contracture, the effects of Ca2+ or D600 were detectable only after a delay of 9 and 18 sec, respectively. It is concluded that divalent cations and D600 compete for the same binding site according to a 1:1 reaction. This site is presumably located inside the transverse tubular system and controls inactivation of the contractile force.

Animals↗

PubMed1994

Functional regulation of the human integrin VLA-1 (CD49a/CD29) by divalent cations and stimulatory beta 1 antibodies.

We have investigated the regulation by divalent cations Mg2+, Ca2+ and Mn2+ of the functional activity of the human integrin VLA-1 expressed on neuroblastoma NB100 cells. VLA-1-mediated adhesion of NB100 cells to ligand collagen type I was supported by either mM concentrations of extracellular Mg2+ or microM levels of Mn2+. In contrast, Ca2+ alone did not induce activation of VLA-1 but exerted a potent inhibitory effect on the Mg(2+)-supported cell adhesion. We have also demonstrated that VLA-1 can be directly activated by the stimulatory monoclonal antibody TS2/16 specific for the integrin beta 1 subunit, resulting in effective adhesion of NB100 cells to type I collagen. This study has been possible by using a novel blocking VLA-alpha 1 specific monoclonal antibody, 5E8D9.

Antibodies, Monoclonal↗

PubMed1983

Studies on the complex formed between bacitracin A and divalent cations.

Bacitracin A is a peptide antibiotic which forms stoichiometric complexes with divalent cations, including Ni2+ and Zn2+. In this paper it is shown that the metal-bacitracin complex contains a group which has a pKa near pH 5.5. Deprotonation of the group is concomitant with the aggregation and precipitation of the metal-bacitracin complex. Bacitracin A, in the absence of metals, does not contain any group which has a pKa in this range. It is postulated that this group is the N-terminal amino of isoleucine, which was previously postulated not to be directly involved in metal coordination based on proton release measurements. An attempt was made to demonstrate directly that the N-terminal amino group is not coordinated to the metal by examining the reactivity of this group with 2,4,6-trinitrobenzene sulfonate. It was clearly shown that bound metals protect the N-terminal amino group from reacting with this reagent. It is speculated that this metal-protection results from a combination of factors, including steric hindrance.

Bacitracin↗

PubMed1982

Effects of divalent cations on the glycolipids from cultured mouse neuroblastoma cells.

The influence of divalent cations on glycosphingolipid metabolism was examined in the NB41A mouse neuroblastoma clonal cell line. HPLC methods were utilized to quantitate the effects on neutral glycolipids and monosialogangliosides. NB41A cells were shown to contain GM3, GM2, GM1, GD3, and GD1a by HPLC and TLC. The neutral glycosphingolipids consisted of glucosylceramide (GlcCer), lactosylceramide (LacCer), GalNAc (beta 1 leads to 4) Gal(beta 1 leads to 4)Glc(beta 1 leads to 1)Cer (GgOse3Cer), and GalNAc(beta 1 leads to 3)Gal(alpha 1 leads to 4) Gal(beta 1 leads to 4)Glc(beta 1 leads to 1)Cer (GbOse4Cer) according to their HPLC behavior. Cells grown in the presence of 1.85 mM-EGTA showed a two-to threefold increase in GM3 whereas other glycosphingolipids were only slightly affected. When cells were grown in the presence of 1.45 mM-EGTA plus 0.4 mM-EDTA a similar increase in GM3 was observed but this change was now accompanied by decreases in GM2, GM1, GgOse3Cer. The EGTA-EDTA effects were reversed when growth was in the presence of Ca2+ sufficient to bind all chelator. Mn2+ replacement reversed the chelator effects differentially; GM2 and GM1 levels were the most sensitive to increases in Mn2+ concentration; GgOse3Cer and GbOse4Cer were also sensitive, whereas GM3 was the least affected. These results suggest calcium serves an important regulatory role on GM3 levels and that manganese concentration may regulate the levels of galactosamine-containing glycolipids in mouse NB41A neuroblastoma cells.

Animals↗

PubMed1987

The effect of divalent cations on the amidolytic activity of bovine plasma activated protein C and des-1-41-light chain activated protein C.

The effect of the divalent cations Ca2+ and Mn2+ on the amidolytic activity of bovine plasma activated protein C and a limited chymotryptic digestion product, des-1-41-light chain activated protein C, which lacks all of the gamma-carboxyglutamic acid present in activated protein C, has been examined at 30 degrees C. In each case, the enzymic activities were dependent upon the presence of these cations, which exerted their effects primarily through influence on the kcat of the reaction. For both enzymes (E), the mechanism of the reaction was most consistent with a rapid equilibrium, random addition of substrate (S), and a single cation (A), with substrate hydrolysis occurring only with the ternary complex of S.E.A. The divalent cation site of importance was not associated with gamma-carboxyglutamic acid residues and was found to be independent of the monovalent cation sites, which also function to activate the amidolytic and esterolytic activities of these enzymes.

Amidohydrolases↗

PubMed1981

Studies on incorporation of membrane protein into liposomes--effect of divalent cations on reconstitution of pig heart mitochondrial H+-ATPase into liposomes.

During the process of reconstitution of pig heart mitochondrial H+-ATPase on liposomes by cholate dialysis method, 1 mM Mg2+ in the dialysis medium can obviously increase activities of the reconstituted H+-ATPase (32Pi-ATP exchange, enzyme activity and its sensitivity to oligomycin or DCCD and ATP-dependent ANS 1) fluorescence). Besides Mg2+, effects of other divalent cations and spermidine on the H+-ATPase reconstitution have been compared. The effectiveness of the divalent cation activation of 32Pi-ATP exchange of the reconstituted H+-ATPase, decreased in the order: Mg2+ greater than Ca2+ greater than Mn2+ greater than Sr2+, while that of increasing the sensitivity to the inhibitors: Ca2+ greater than Mg2+ greater than Mn2+ greater than Sr2+. No significant effect on the H+-ATPase reconstitution has been found with Cd2+, Zn2+ and spermidine3+. Mechanism of action of the divalent cations on reconstitution has been discussed.

Adenosine Triphosphatases↗

PubMed2002

Modulation of rod photoreceptor potassium Kx current by divalent cations.

Similarities between photoreceptor Kx channels and EAG and KCNQ channels suggest that the Kx channel may be related to one or both of these families of potassium channels. To provide a basis for further comparison of these channels, as well as to reveal the role of zinc (endogenously present in photoreceptors) in Kx channel modulation, we studied the effects of divalent cations on activation and kinetic properties of the noninactivating potassium current, I(Kx), in salamander rod photoreceptors. We found that divalent cations slowed the activation of I(Kx) and shifted its activation curve to positive potentials, effects attributable to surface charge screening. The effect of zinc on I(Kx) kinetics was considerably stronger than that expected from the activation curve shift alone. None of the divalent ions studied changed the monoexponential nature of I(Kx) activation. A simple model of a rod photoreceptor was created using NEURON software. In the model, zinc accelerated and sharpened the light response, broadening the frequency response of rod photoreceptors. Our results show that although the overall effects of divalent cations are in part due to surface charge screening effects, zinc also induced a specific change in I(Kx) activation kinetics, and this action profoundly alters rod response properties.

Ambystoma↗

PubMed1972

Effect of divalent cations and pH on intrinsic factor-mediated attachment of vitamin B 12 to intestinal microvillous membranes.

Calcium, but not other divalent cations, is required for optimal uptake of intrinsic factor-bound (57)Co-labeled cyanocobalamin (IFB(12)) by microvillous membranes isolated from hamster ileal-absorptive cells. Chelation of divalent cations by disodium ethylenediaminetetraacetate (EDTA) promptly removes IFB(12) previously attached to microvillous membranes. High concentrations of CaCl(2) or MgCl(2) also markedly inhibit membrane uptake of IFB(12) and rapidly remove previously attached IFB(12). Similarly, reduction of pH to below 5.4 prevents membrane attachment of IFB(12) and removes virtually all IFB(12) already bound to microvillous membranes. The effects of calcium depletion, increased salt concentrations, and acidification on membrane uptake of IFB(12) were completely reversible. These findings are consistent with the concept that the formation of calcium salt bridges is essential for attachment of IFB(12) to the ileal-absorptive surface.

Animals↗

PubMed1997

Effects of divalent cations on M-cadherin expression and distribution during primary rat myogenesis in vitro.

In the process of myogenesis, cadherins are thought to be involved in the initial cell-cell recognition and possible initiation of myoblast fusion to form multinucleated myotubes. Of the cadherins, M-cadherin, but not N-cadherin, is down-regulated upon inhibition of myogenesis, suggesting that M-cadherin may be a key receptor involved in myogenesis. M-cadherin binds in a calcium-dependent manner, and depletion of divalent cations inhibits myoblast fusion. We analyzed the regulation of M-cadherin protein and mRNA levels in primary rat myogenic cultures in the presence and absence of divalent cations. In untreated cultures M-cadherin was localized to various myogenic cell-cell contacts. M-cadherin protein and mRNA levels showed a peak at day 2 after the initiation of growth. When divalent cations were removed from the cell culture medium, myoblast fusion was inhibited and immunocytochemical analysis revealed a failure of M-cadherin to localize to cell-cell contacts. Analysis of M-cadherin protein and mRNA in fusion-inhibited cultures still revealed a peak at day 2. However, by day 3, M-cadherin protein levels in the fusion-inhibited cultures were reduced in both the detergent-soluble and -insoluble fractions in comparison with the untreated cultures. Interestingly, beta-catenin, a protein associated with cadherins, was frequently observed at intercellular contacts in the fusion-inhibited cultures. We could also show that the intracellular levels of beta-catenin protein remained constant regardless of the presence or absence of divalent cations. In summary, the dynamic regulation of M-cadherin in muscle-fusion-related events is an indication of the importance of M-cadherin for myoblast fusion and myogenic differentiation.

Animals↗

PubMed2005

Effects of divalent cations on drug encapsulation efficiency of deacylated gellan gum.

The objective of this research was to evaluate the effects of various divalent cations on the encapsulation efficiency of gellan gum and to probe the underlying mechanisms responsible for drug-loading efficiency. Spherical beads containing azathioprine were prepared from deacetylated gellan gum by ionotropic gelation method. One molar solution of various divalent chlorides (MgCl(2), BaCl(2), CaCl(2), CuCl(2) and ZnCl(2)) and two additional concentrations of CaCl(2) (2.5 M and 5.0 M) were used as ionotropic media. Drug solubility was also determined in these ionotropic media and statistically evaluated using ANOVA. Solubility in various divalent chloride solutions (1.0 M) suggests that azathioprine forms complex with Ca(2+), Zn(2+) and Cu(2+), while there might be a formation of poorly water-soluble chelates with Mg(2+) and Ba(2+) as solubility in these media were less than in deionized water. The encapsulation efficiency of gellan gum was much higher in the presence of transition elements (Cu(2+) and Zn(2+)) when compared to alkaline earth metal ions (Ca(2+), Mg(2+) and Ba(2+)). Higher concentrations of Ca(2+) decreased the encapsulation efficiency of gellan gum in a nearly proportional manner. The correlation between encapsulation efficiency and pH of the ionotropic media was negative and significant (r=-0.9574, p<0.05), although the solubility of azathioprine seems to be independent of the pH of the ionotropic medium. Overall, the results suggest that drug encapsulation efficiency of deacetylated gellan gum is largely affected by the concentration and nature of various divalent cations (e.g. atomic number, valency or electro-positivity, co-ordination property, etc.) and pH of the ionotropic medium.

Antimetabolites↗