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Adiabatic differential scanning calorimetric study of divalent cation induced DNA - DPPC liposome formulation compacted for gene delivery

Süleymanoglu,Erhan
Fonte: Instituto de Tecnologia do Paraná - Tecpar Publicador: Instituto de Tecnologia do Paraná - Tecpar
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/11/2004 EN
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56.45%
Complexes between nucleic acids and phospholipid vesicles have been developed as stable non-viral gene delivery vehicles. Currently employed approach uses positively charged lipid species and a helper zwitterionic lipid, the latter being applied for the stabilization of the whole complex. However, besides problematic steps during their preparation, cationic lipids are toxic for cells. The present work describes some energetic issues pertinent to preparation and use of neutral lipid-DNA self-assemblies, thus avoiding toxicity of lipoplexes. Differential scanning calorimetry data showed stabilization of polynucleotide helix upon its interaction with liposomes in the presence of divalent metal cations. It is thus possible to suggest this self-assembly as an improved formulation for use in gene delivery.

Binding of a single divalent cation directly correlates with the blue-to-purple transition in bacteriorhodopsin.

Jonas, R; Ebrey, T G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/01/1991 EN
Relevância na Pesquisa
46.65%
We have characterized a unique divalent cation binding site on bacteriorhodopsin which controls the blue-to-purple transition in the purple membrane of Halobacterium halobium. To identify this site we first showed the correlation between the binding of one Ca2+ per bacteriorhodopsin and the amount of blue membrane converted to purple membrane. When the free Ca2+ was reduced below 1 microM, and the pH was set below 5.0 with 0.5 mM citrate, only binding to this high-affinity site was observed, and we could separate its effect from the effect of other divalent cations binding to the membrane under other conditions. Second, the titration of purple membrane showed that protons are taken up in two distinct steps, about 13 with a pKa of 4-5 and an additional 2 protons with a pKa of 2.75, in 5 mM MgSO4. The latter is identical to the pKa for the purple-to-blue transition in 5 mM MgSO4. Taken together, these observations strongly suggest a direct role for cations in the regulation of the bacteriorhodopsin color under normal conditions. We have also found that the intrinsic pKa for the purple-to-blue transition is about 2.05, suggesting this is the pKa of the group or groups that, when protonated, lead to the blue membrane. Previously published data can now be interpreted to suggest that the cation regulates an active site near the retinal chromophore. A binding site for the divalent cation that includes Asp-212 and interactions with the protonated Schiff base...

The integral divalent cation within the intermolecular purine*purine. pyrimidine structure: a variable determinant of the potential for and characteristics of the triple helical association.

Blume, S W; Lebowitz, J; Zacharias, W; Guarcello, V; Mayfield, C A; Ebbinghaus, S W; Bates, P; Jones, D E; Trent, J; Vigneswaran, N; Miller, D M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/01/1999 EN
Relevância na Pesquisa
46.85%
In vitro assembly of an intermolecular purine*purine.pyrimidine triple helix requires the presence of a divalent cation. The relationships between cation coordination and triplex assembly were investigated, and we have obtained new evidence for at least three functionally distinct potential modes of divalent cation coordination. (i) The positive influence of the divalent cation on the affinity of the third strand for its specific target correlates with affinity of the cation for coordination to phosphate. (ii) Once assembled, the integrity of the triple helical structure remains dependent upon its divalent cation component. A mode of heterocyclic coordination/chelation is favorable to triplex formation by decreasing the relative tendency for efflux of integral cations from within the triple helical structure. (iii) There is also a detrimental mode of base coordination through which a divalent cation may actively antagonize triplex assembly, even in the presence of other supportive divalent cations. These results demonstrate the considerable impact of the cationic component, and suggest ways in which the triple helical association might be positively or negatively modulated.

Control of tumbling in bacterial chemotaxis by divalent cation.

Ordal, G W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1976 EN
Relevância na Pesquisa
46.73%
Chemotaxis is migration of organisms to higher concentrations of attractant or lower concentrations of repellent. Understanding the switch than controls whether the flagella rotate counterclockwise for swimming or clockwise for tumbling (thrashing about without making much forward progress) is central to understanding chemotaxis of peritrichous bacteria, since chemotaxis results from selective suppression of tumbles. Depletion of divalent cation by chelating agents in the presence of A23187, an ionophore that conveys divalent cation across membrane, causes incessant tumbling in Bacillus subtilis. Small additions of MgCl2 prevent this tumbling. In this tumbling condition, the bacteria which normally swim extensively when given attractant, do not respond even to 10 mM alanine, a strong attractant. MnCl2, by contrast to others potentiated by the ionophore. Permanent cations, including tetraphenylarsonium ion and triphenylmethylphosphonium ion, cause permanent swimming, even in the presence of A23187 and chelating agents. We propose that divalent cation, probably Mg2+ ion, binds to the switch to cause swimming and that, in the absence of divalent cation at the switch, the bacterium tumbles.

Divalent Cation Activation of Deoxycholate-Solubilized and -Inactivated Membrane Reduced Nicotinamide Adenine Dinucleotide Oxidase of Bacillus megaterium KM1

Eisenberg, R. C.; Yu, L.; Wolin, M. J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1970 EN
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46.7%
After treating Bacillus megaterium KM membranes with 0.2% sodium deoxycholate, most of the membrane reduced nicotinamide adenine dinucleotide (NADH) oxidase was inactivated, and all of the membrane NADH-2,6 dichlorophenol indophenol oxidoreductase was solubilized. Dilution of the deoxycholate-treated membranes in the presence of divalent cations restored almost all of the original membrane NADH oxidase. The effectiveness of the divalent cation activation decreased in the order Ba2+ > Ca2+ > Mg2+ > Mn2+. After centrifugation, the deoxycholate-treated membranes at 100,000 × g for 1 hr, all of the NADH oxidase that was activated by a divalent cation was soluble. Cation-activated oxidase, however, was insoluble. The results show that 0.2% deoxycholate at least partially solubilizes the total electron chain from NADH to O2 in an inactive from which can be reactivated by divalent cations with the formation of active, insoluble NADH oxidase.

Locus of Divalent Cation Inhibition of the Bactericidal Action of Polymyxin B

Chen, Chuen-Chin Hsu; Feingold, David S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1972 EN
Relevância na Pesquisa
46.64%
The bactericidal effect of polymyxin B upon two Pseudomonas aeruginosa strains and two Escherichia coli strains was studied as a function of the concentration of the divalent cations Ca2+ and Mg2+. Lipid spherules in aqueous suspension (liposomes) were prepared from the bacterial phospholipids and were examined for the release of trapped glucose induced by polymyxin B under similar conditions of divalent cation concentration. In contrast to the bacteria, which were protected markedly from the bactericidal action of polymyxin B by Ca2+ or Mg2+ at concentrations of 2 × 10−3m or less, neither cation had any protective effect on bacterial liposomes at concentrations up to 2 × 10−2m. These observations suggest that divalent cations antagonize the bactericidal effect of polymyxin B indirectly through interaction with the cell wall rather than at the primary membrane locus of action. The interaction of the divalent cations with the cell wall prevents access of the antibiotic to the cytoplasmic membrane by mechanisms that remain unclear.

Tetrodotoxin-resistant sodium current of rat nodose neurones: monovalent cation selectivity and divalent cation block.

Ikeda, S R; Schofield, G G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1987 EN
Relevância na Pesquisa
46.67%
1. Monovalent cation selectivity and divalent cation sensitivity of the tetrodotoxin (TTX)-resistant Na+ current in dissociated adult rat nodose ganglion neurones were investigated using the whole-cell patch-clamp technique. 2. The TTX-resistant Na+ current was isolated using ion substitution and pharmacological agents. Under these conditions, the current reversal potential shifted 52 mV per tenfold change in external [Na+]. 3. Inorganic and organic monovalent cation permeability ratios (Px/PNa) were determined from changes in reversal potential and the Goldman-Hodgkin-Katz equation. The Px/PNa values determined by the former method were HONH3+, 1.38; Li+, 1.00; H2NNH3+, 0.66; NH4+, 0.28; CH3NH3+, less than 0.13; K+, less than 0.13; Rb+, less than 0.12; Cs+, less than 0.10; (CH3)4N+, less than 0.10. The values determined by either method agreed within 10%. 4. The effects of Cd2+, Co2+, Mn2+ and Ni2+ on the TTX-resistant Na+ current were analysed from peak-conductance values. These ions shifted the activation of the current to more positive potentials and decreased the maximal conductance. At 3 mM concentrations, Cd2+, Ni2+, Co2+ and Mn2+ decreased the maximal conductance 64.6, 50.7, 25.0 and 20.3%, respectively. 5. The results indicate that: (a) the monovalent cation selectivity of the TTX-resistant Na+ current is similar to that of the TTX-sensitive Na+ current in other tissues; and (b) the TTX-resistant Na+ current is less sensitive to divalent cations than the Ca2+ current in these neurones. These observations suggest that the structure determining the monovalent cation permeability of the TTX-resistant Na+ current is similar to that of the TTX-sensitive Na+ current in other tissues...

Localization of divalent cation-binding site in the pore of a small conductance Ca(2+)-activated K(+) channel and its role in determining current-voltage relationship.

Soh, Heun; Park, Chul-Seung
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/2002 EN
Relevância na Pesquisa
46.64%
In our previous study, we proposed that the inwardly rectifying current-voltage (I-V) relationship of small-conductance Ca(2+)-activated K(+) channels (SK(Ca) channels) is the result of voltage-dependent blockade of K(+) currents by intracellular divalent cations. We expressed a cloned SK(Ca) channel, rSK2, in Xenopus oocytes and further characterized the nature of the divalent cation-binding site by electrophysiological means. Using site-directed substitution of hydrophilic residues in K(+)-conducting pathway and subsequent functional analysis of mutations, we identified an amino acid residue, Ser-359, in the pore-forming region of rSK2 critical for the strong rectification of the I-V relationship. This residue interacts directly with intracellular divalent cations and determines the ionic selectivity. Therefore, we confirmed our proposition by localizing the divalent cation-binding site within the conduction pathway of the SK(Ca) channel. Because the Ser residue unique for the subfamily of SK(Ca) channels is likely to locate closely to the selectivity filter of the channels, it may also contribute to other permeation characteristics of SK(Ca) channels.

Fluorescence study of the divalent cation-transport mechanism of ionophore A23187 in phospholipid membranes.

Kolber, M A; Haynes, D H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1981 EN
Relevância na Pesquisa
46.77%
The mechanism for transport of divalent cations across phospholipid bilayers by the ionophore A23187 was investigated. The intrinsic fluorescence of the ionophore was used in equilibrium and rapid-mixing experiments as an indicator of ionophore environment and complexation with divalent cations. The neutral (protonated) form of the ionophore binds strongly to the membrane, with a high quantum yield relative to that in the aqueous phase. The negatively charged form of the ionophore binds somewhat less strongly, with a lower quantum yield, and does not move across the membrane. Complexation of the negatively charged form with divalent cations was measured by the decrease in fluorescence. An apparent rate constant (kapp) for transport of the ionophore across the membrane was determined from the rate of fluorescence changes observed in stopped-flow rapid kinetic experiments. The variation of kapp was studied as a function of pH, temperature, ionophore concentration, membrane lipid composition, and divalent cation concentration and type. Analysis and comparison with equilibrium constants for protonation and complexation show that A23187 and its metal:ionophore complexes bind near the membrane-water interface in the lipid polar-head region. The interfacial reactions occur rapidly...

Divalent cation dependence of the inhibition by phenothiazines of mediator release from mast cells.

Peachell, P. T.; Pearce, F. L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1989 EN
Relevância na Pesquisa
46.67%
1. The divalent cations calcium, strontium and barium--and in that order of decreasing effectiveness--were capable of supporting the stimulated release of histamine from rat peritoneal mast cells (RPMC). 2. The responsiveness of mast cells to stimulation in the presence of divalent cations was, in general, markedly enhanced when the cells were first depleted of their intracellular calcium stores. 3. The putative calmodulin antagonists, chlorpromazine, promethazine, thioridazine (phenothiazines) and W-7 (a naphthalene sulphonamide) all inhibited histamine release in the presence of divalent cations in both untreated cells and in RPMC depleted of their intracellular calcium. 4. Histamine release induced by antigen, compound 48/80 and ionophore A23187 was inhibited by this class of compounds most effectively in the presence of extracellular barium, less so in the presence of strontium and least so in calcium-containing media. 5. In the experimental situation where the extracellular calcium concentration was reduced (less than 1 mM), the phenothiazines inhibited the stimulated release of histamine more effectively. 6. In toto, these results suggest that strontium and barium, as well as calcium, can support histamine release from RPMC by directly interacting with an intracellular divalent cation-binding site that may be calmodulin. As a consequence...

Mutation of putative divalent cation sites in the alpha 4 subunit of the integrin VLA-4: distinct effects on adhesion to CS1/fibronectin, VCAM-1, and invasin

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/10/1993 EN
Relevância na Pesquisa
46.86%
To investigate the functional significance of putative integrin divalent cation binding sites, several mutated alpha 4 subunit cDNAs were constructed. Mutants contained the conservative substitution of Glu for Asp or Asn at the third position in each of three putative divalent cation sites. Transfection of wild-type or mutated alpha 4 into K562 cells yielded comparable expression levels and immunoprecipitation profiles. However, for all three alpha 4 mutants, adhesion to CS1/fibronectin was greatly diminished in either the presence or absence of the stimulatory anti-beta 1 mAb TS2/16. Constitutive adhesion to vascular cell adhesion molecule (VCAM) 1 was also diminished but, unlike CS1 adhesion, was restored upon TS2/16 stimulation. In contrast, adhesion to the bacterial protein invasin was minimally affected by any of the three mutations. For each of the mutants, the order of preference for divalent cations was unchanged compared to wild-type alpha 4, on CS1/fibronectin (Mn2+ > Mg2+ > Ca2+), on VCAM-1 (Mn2+ > Mg2+ = Ca2+) and on invasin (Mg2+ = Ca2+). However for the three mutants, the efficiency of divalent cation utilization was decreased. On VCAM-1, 68-108 microM Mn2+ was required to support half-maximal adhesion for the mutants compared with 14-18 microM for wild-type alpha 4. These results indicate (a) that three different ligands for VLA-4 show widely differing sensitivities to mutations within putative divalent cation sites...

Chemical properties of the divalent cation binding site on potassium channels

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/08/1992 EN
Relevância na Pesquisa
46.7%
The actions of divalent cations on voltage-gated ion channels suggest that these cations bind to specific sites and directly influence gating kinetics. We have examined some chemical properties of the external divalent cation binding sites on neuronal potassium channels. Patch clamp techniques were used to measure the electrophysiological properties of these channels and Zn ions were used to probe the divalent cation binding site. The channel activation kinetics were greatly (three- to fourfold) slowed by low (2-5 mM) concentrations of Zn; deactivation kinetics were only slightly affected. These effects of Zn were inhibited by low solution pH in a manner consistent with competition between Zn and H ions for a single site. The apparent inhibitory pK for this site was near 7.2. Treatment of the neurons with specific amino acid reagents implicated amino, but no histidyl or sulfhydryl, residues in divalent cation binding.

Divalent Cation Sensitivity of BK Channel Activation Supports the Existence of Three Distinct Binding Sites

Zeng, Xu-Hui; Xia, Xiao-Ming; Lingle, Christopher J.
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em /03/2005 EN
Relevância na Pesquisa
46.81%
Mutational analyses have suggested that BK channels are regulated by three distinct divalent cation-dependent regulatory mechanisms arising from the cytosolic COOH terminus of the pore-forming α subunit. Two mechanisms account for physiological regulation of BK channels by μM Ca2+. The third may mediate physiological regulation by mM Mg2+. Mutation of five aspartate residues (5D5N) within the so-called Ca2+ bowl removes a portion of a higher affinity Ca2+ dependence, while mutation of D362A/D367A in the first RCK domain also removes some higher affinity Ca2+ dependence. Together, 5D5N and D362A/D367A remove all effects of Ca2+ up through 1 mM while E399A removes a portion of low affinity regulation by Ca2+/Mg2+. If each proposed regulatory effect involves a distinct divalent cation binding site, the divalent cation selectivity of the actual site that defines each mechanism might differ. By examination of the ability of various divalent cations to activate currents in constructs with mutationally altered regulatory mechanisms, here we show that each putative regulatory mechanism exhibits a unique sensitivity to divalent cations. Regulation mediated by the Ca2+ bowl can be activated by Ca2+ and Sr2+, while regulation defined by D362/D367 can be activated by Ca2+...

Acidic pH and divalent cation sensing by PhoQ are dispensable for systemic salmonellae virulence

Hicks, Kevin G; Delbecq, Scott P; Sancho-Vaello, Enea; Blanc, Marie-Pierre; Dove, Katja K; Prost, Lynne R; Daley, Margaret E; Zeth, Kornelius; Klevit, Rachel E; Miller, Samuel I
Fonte: eLife Sciences Publications, Ltd Publicador: eLife Sciences Publications, Ltd
Tipo: Artigo de Revista Científica
Publicado em 23/05/2015 EN
Relevância na Pesquisa
46.64%
Salmonella PhoQ is a histidine kinase with a periplasmic sensor domain (PD) that promotes virulence by detecting the macrophage phagosome. PhoQ activity is repressed by divalent cations and induced in environments of acidic pH, limited divalent cations, and cationic antimicrobial peptides (CAMP). Previously, it was unclear which signals are sensed by salmonellae to promote PhoQ-mediated virulence. We defined conformational changes produced in the PhoQ PD on exposure to acidic pH that indicate structural flexibility is induced in α-helices 4 and 5, suggesting this region contributes to pH sensing. Therefore, we engineered a disulfide bond between W104C and A128C in the PhoQ PD that restrains conformational flexibility in α-helices 4 and 5. PhoQW104C-A128C is responsive to CAMP, but is inhibited for activation by acidic pH and divalent cation limitation. phoQW104C-A128C Salmonella enterica Typhimurium is virulent in mice, indicating that acidic pH and divalent cation sensing by PhoQ are dispensable for virulence.

Nucleotide and divalent cation specificity of in vitro iron-molybdenum cofactor synthesis.

Chatterjee, R; Allen, R M; Shah, V K; Ludden, P W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1994 EN
Relevância na Pesquisa
46.65%
The nucleotide and divalent cation requirements of the in vitro iron-molybdenum cofactor (FeMo-co) synthesis system have been compared with those of substrate reduction by nitrogenase. The FeMo-co synthesis system specifically requires ATP, whereas both 1,N6-etheno-ATP and 2'-deoxy-ATP function in place of ATP in substrate reduction (M. F. Weston, S. Kotake, and L. C. Davis, Arch. Biochem. Biophys. 225:809-817, 1983). Mn2+, Ca2+, and Fe2+ substitute for Mg2+ to various extents in in vitro FeMo-co synthesis, whereas Ca2+ is ineffective in substrate reduction by nitrogenase. The observed differences in the nucleotide and divalent cation specificities suggest a role(s) for the nucleotide and divalent cation in in vitro FeMo-co synthesis that is distinct from their role(s) in substrate reduction.

Mechanism and Role of Divalent Cation Binding of Bacteriorhodopsin

Chang, C. -H.; Jonas, R.; Melchiore, S.; Govindjee, R.; Ebrey, T. G.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1986 EN
Relevância na Pesquisa
46.64%
Several observations have already suggested that the carboxyl groups are involved in the association of divalent cations with bacteriorhodopsin (Chang et al., 1985). Here we show that at least part of the protons released from deionized purple membrane (`blue membrane') samples when salt is added are from carboxyl groups. We find that the apparent pK of magnesium binding to purple membrane in the presence of 0.5 mM buffer is 5.85. We suggest this is the pK of the carboxyl groups shifted from their usual pK because of the proton concentrating effect of the large negative surface potential of the purple membrane. Divalent cations may interact with negatively charged sites on the surface of purple membrane through the surface potential and/or through binding either by individual ligands or by conformation-dependent chelation. We find that divalent cations can be released from purple membrane by raising the temperature. Moreover, purple membrane binds only about half as many divalent cations after bleaching. Neither of these operations is expected to decrease the surface potential and thus these experiments suggest that some specific conformation in purple membrane is essential for the binding of a substantial fraction of the divalent cations. Divalent cations in purple membrane can be replaced by monovalent...

Differential divalent cation requirements uncouple the assembly and catalytic reactions of human immunodeficiency virus type 1 integrase.

Hazuda, D J; Felock, P J; Hastings, J C; Pramanik, B; Wolfe, A L
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1997 EN
Relevância na Pesquisa
46.73%
Previous in vitro analyses have shown that the human immunodeficiency virus type 1 (HIV-1) integrase uses either manganese or magnesium to assemble as a stable complex on the donor substrate and to catalyze strand transfer. We now demonstrate that subsequent to assembly, catalysis of both 3' end processing and strand transfer requires a divalent cation cofactor and that the divalent cation requirements for assembly and catalysis can be functionally distinguished based on the ability to utilize calcium and cobalt, respectively. The different divalent cation requirements manifest by these processes are exploited to uncouple assembly and catalysis, thus staging the reaction. Staged 3' end processing and strand transfer assays are then used in conjunction with exonuclease III protection analysis to investigate the effects of integrase inhibitors on each step in the reaction. Analysis of a series of related inhibitors demonstrates that these types of compounds affect assembly and not either catalytic process, therefore reconciling the apparent disparate results obtained for such inhibitors in assays using isolated preintegration complexes. These studies provide evidence for a distinct role of the divalent cation cofactor in assembly and catalysis and have implications for both the identification and characterization of integrase inhibitors.

Divalent cation and ionic strength effects on Vinca alkaloid-induced tubulin self-association.

Lobert, S; Boyd, C A; Correia, J J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1997 EN
Relevância na Pesquisa
46.73%
We present here a systematic study of ionic strength and divalent cation effects on Vinca alkaloid-induced tubulin spiral formation. We used sedimentation velocity experiments and quantitative fitting of weight-average sedimentation coefficients versus free drug concentrations to obtain thermodynamic parameters under various solution conditions. The addition of 50-150 mM NaCl to our standard buffer (10 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM Mg, 50 microM GDP or GTP, pH 6.9) enhances overall vinblastine- or vincristine-induced tubulin self-association. As demonstrated in previous studies, GDP enhances overall self-association more than GTP, although in the presence of salt, GDP enhancement is reduced. For example, in 150 mM NaCl, GDP enhancement is 0.24 kcal/mol for vinblastine and 0.36 kcal/mol for vincristine versus an average enhancement of 0.87 (+/- 0.34) kcal/mol for the same drugs in the absence of salt. Wyman linkage analysis of experiments with vinblastine or vincristine over a range of NaCl concentrations showed a twofold increase in the change in NaCl bound to drug-induced spirals in the presence of GTP compared to GDP. These data indicate that GDP enhancement of Vinca alkaloid-induced tubulin self-association is due in part to electrostatic inhibition in the GTP state. In the absence of NaCl...

Divalent cation tolerance protein binds to β-secretase and inhibits the processing of amyloid precursor protein☆

Liu, Runzhong; Hou, Haibo; Yi, Xuelian; Wu, Shanwen; Zeng, Huan
Fonte: Medknow Publications & Media Pvt Ltd Publicador: Medknow Publications & Media Pvt Ltd
Tipo: Artigo de Revista Científica
Publicado em 15/04/2013 EN
Relevância na Pesquisa
46.84%
The deposition of amyloid-beta is a pathological hallmark of Alzheimer's disease. Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase (beta-site amyloid precursor protein-cleaving enzyme 1) and γ-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of Alzheimer's disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1-interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment...

Divalent cation competition with [3H]saxitoxin binding to tetrodotoxin- resistant and -sensitive sodium channels. A two-site structural model of ion/toxin interaction

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/02/1993 EN
Relevância na Pesquisa
46.73%
Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from their binding sites on nerve and skeletal muscle Na channels. Recent studies of cloned cardiac (toxin-resistant) and brain (toxin-sensitive) Na channels suggest important structural differences in their toxin and divalent cation binding sites. We used a partially purified preparation of sheep cardiac Na channels to compare monovalent and divalent cation competition and pH dependence of binding of [3H]STX between these toxin-resistant channels and toxin-sensitive channels in membranes prepared from rat brain. The effects of several chemical modifiers of amino acid groups were also compared. Toxin competition curves for Na+ in heart and Cd2+ in brain yielded similar KD values to measurements of equilibrium binding curves. The monovalent cation sequence for effectiveness of [3H]STX competition is the same for cardiac and brain Na channels, with similar KI values for each ion and slopes of -1. The effectiveness sequence corresponds to unhydrated ion radii. For seven divalent cations tested (Ca2+, Mg2+, Mn2+, Co2+, Ni2+, Cd2+, and Zn2+) the sequence for [3H]STX competition was also similar. However, whereas all ions displaced [3H]STX from cardiac Na channels at lower concentrations...