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At least 433 records · Page 24Linked to original sources
PubMed1983

Stabilization of glucocorticoid receptors in vitro using divalent cations plus cation chelators.

The specific glucocorticoid receptor binding of rat liver cytosol was very unstable in vitro at 25 and 4 degrees C. However, 5 mM CaCl2 added with 5 mM EDTA to cytosol prior to incubation markedly stabilized unbound glucocorticoid receptors at both temperatures. Optimal effectiveness was achieved using equimolar (5 mM) amounts of CaCl2 and EDTA. On the other hand, 5 mM CaCl2 (added alone) further destabilized the unbound glucocorticoid receptor, while 5 mM EDTA (added alone) had no effect at 25 degrees C. EGTA (in lieu of EDTA) added with CaCl2 stabilized hepatic receptor binding at 25 degrees C. On the other hand, citrate added with calcium was ineffective in stabilizing the hepatic glucocorticoid receptor. MgCl2 effectively replaced CaCl2 as a stabilizing agent at 25 degrees C if added with 5 mM EDTA. When added alone, MgCl2 slightly destabilized the unbound receptor. Sucrose density gradient analysis (in low salt) revealed that CaCl2 plus EDTA enhanced the steroid-receptor complex sedimentation coefficient from 7 S to about 10 S. Unlike molybdate, CaCl2 plus EDTA had no apparent effect on steroid-receptor complex thermal transformation into a nuclear binding form, while MgCl2 plus EDTA partially reduced transformation. These results suggest a novel means to chemically stabilize unbound hepatic glucocorticoid receptors in vitro which may be of particular importance for receptor purification studies.

Animals↗
PubMed1987

Stabilization of phosphofructokinase with sugars during freeze-drying: characterization of enhanced protection in the presence of divalent cations.

Phosphofructokinase purified from rabbit skeletal muscle is fully inactivated after freeze-drying and dissolution. The addition of trehalose or maltose to the enzyme solution prior to freeze-drying results in a recovery of up to 80% of the original activity. Slightly less stabilization is imparted by sucrose, whereas glucose and galactose at concentrations up to 500 mM are relatively ineffective at protecting phosphofructokinase. Addition of ionic zinc to enzyme-sugar mixtures prior to freeze-drying greatly enhances the stabilization imparted by the above sugars. This effect is not simply due to the summation of the individual protective capacities of zinc and the sugar. Zinc alone affords no protection, but a high degree of stabilization is achieved when zinc is added to a sugar solution, even when the sugar is at a concentration at which, by itself, it is totally ineffective. In the presence of a constant sugar concentration (100 mM), freeze-dry stabilization of phosphofructokinase is increased as the concentration of zinc is increased. When the zinc concentration is held constant (0.9 mM) and the sugar concentration varied, the maximum stabilization is noted with less than 200 mM sugar. At higher solute concentrations the degree of enhancement decreases such that with 500 mM sugar the addition of zinc results in only a slight increase in protection. Several other organic solutes (proline, 4-hydroxyproline, glycine, trimethylamine N-oxide, glycerol and myo-inositol) that afford cryoprotection to phosphofructokinase, an effect enhanced by the addition of zinc, do not stabilize the enzyme during freeze-drying, even if zinc is present. The addition of ionic copper, cadmium, nickel, cobalt, calcium and manganese to trehalose-phosphofructokinase solutions prior to freeze-drying also increases the percentage of activity recovered after dissolution. Magnesium is ineffective in this respect.

Animals↗
PubMed1989

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.

Cations, Divalent↗
PubMed1991

Effects of divalent cations on the time course of post-tetanic decay of miniature endplate potential frequency in frogs.

Relatively high external Mg2+ specifically induces a shoulder in the post-tetanic decay of miniature endplate potential frequency at frog neuromuscular junctions. This effect is antagonized by Ca2+, but not by Sr2+ and Mn2+. This shoulder formation is not caused by Mn2+ or dinitrophenol in the medium. It is suggested that Mg2+ enters nerve terminals and displaces Ca2+ from internal stores; Mg2+ subsequently interferes with Ca2+ removal until the Mg2+ itself has been removed. The dinitrophenol result suggests that ATP-dependent Ca2+ extrusion is not permanently slowed. An equally likely and simpler explanation is that Mg2+ enters nerve terminals and the Mg2+ itself causes an increase in miniature endplate potential frequency. External Ca2+ competes with Mg2+ for channel entry and prevents this effect. The time course of the decay of miniature endplate potential frequency reflects the processes involved in Mg2+ extrusion and/or uptake.

2,4-Dinitrophenol↗
PubMed1994

Effect of divalent cations on hemolysin synthesis by Serpulina (Treponema) hyodysenteriae: inhibition induced by zinc and copper.

Serpulina hyodysenteriae produces an oxygen-stable heat-labile hemolysin that may be an important virulence factor in the pathogenesis of swine dysentery. We examined the effect of Ca2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, Ni2+, and Zn2+ on the hemolytic activity of cell-free supernatant (CFS) from S. hyodysenteriae, isolate B204. Cells harvested from late logarithmic phase cultures were incubated in phosphate-buffered saline containing glucose and RNA-core (PBS-GR) with or without cations and the hemolytic activity of CFS obtained after successive 30 min incubation and washing cycles was determined. The addition of either ZnSO4 or CuSO4 to the PBS-GR caused complete inhibition of hemolytic activity after 3 cycles; other cations gave results similar to control extracts. Reduction in the concentration of Zn2+ in CFS by 60 to 80% after each incubation cycle and binding of Zn2+ by EDTA indicated that Zn2+ was associated with the cell fraction, and inhibition of hemolysin activity was specifically mediated by Zn2+. When the spirochetes were washed after incubation in the presence of ZnSO4 for 2 cycles and incubated in fresh PBS-GR without Zn2+, inhibition of hemolysin activity remained unchanged, indicating that the inhibitory effect of ZnSO4 was due to a direct action of ZnSO4 on the spirochetes. Since neither the viability of the spirochetes nor the activity of pre-formed hemolysin were affected by the presence of ZnSO4, the inhibitory effect of Zn2+ cations was attributed to reduced biosynthesis by viable S. hyodysenteriae cells rather than interference of Zn2+ cations with lysis of erythrocytes by the hemolysin. Transmission electron microscopic examination of spirochetes after incubation in PBS-GR containing ZnSO4 revealed clumping of ribosomes and clearing of cell cytoplasm.

Animals↗
PubMed1979

X-ray diffraction study of the kinetics of myelin lattice swelling. Effect of divalent cations.

The time-course of myelin lattice swelling and its reversal in dissected peripheral nerves was determined by small-angle x-ray diffraction using a position-sensitive proportional detector. The process of swelling can take place either in several hours or in less than 1 h depending on pretreatment of the nerves. The reversal of swelling was always completed within 1 h. The rapid structural transitions involved the disordering of membrane pairs as indicated by the transient appearance of a continuous intensity distribution similar to the membrane pair transform for myelin. The slow transitions involved the gradual replacement of the discrete reflections from the native structure by the reflections from the swollen lattice. Myelin membrane arrays reformed in normal Ringer's solution were much more stable to subsequent swelling than arrays reformed in Ca+2 and Mg+2-free Ringer's. These results suggest that these ions participate in stabilizing the interactions between the external surfaces of adjacent membrane pairs.

Animals↗
PubMed1997

Spectroscopic study of conformational changes in subdomain 1 of G-actin: influence of divalent cations.

Temperature dependence of the fluorescence intensity and anisotropy decay of N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to Cys374 of actin monomer was investigated to characterize conformational differences between Ca- and Mg-G-actin. The fluorescence lifetime is longer in Mg-G-actin than that in Ca-G-actin in the temperature range of 5-34 degrees C. The width of the lifetime distribution is smaller by 30% in Mg-saturated actin monomer at 5 degrees C, and the difference becomes negligible above 30 degrees C. The semiangle of the cone within which the fluorophore can rotate is larger in Ca-G-actin at all temperatures. Electron paramagnetic resonance measurements on maleimide spin-labeled (on Cys374) monomer actin gave evidence that exchange of Ca2+ for Mg2+ induced a rapid decrease in the mobility of the label immediately after the addition of Mg2+. These results suggest that the C-terminal region of the monomer becomes more rigid as a result of the replacement of Ca2+ by Mg2+. The change can be related to the difference between the polymerization abilities of the two forms of G-actin.

Actins↗
PubMed1998

The influence of divalent cations on the dynamic properties of actin filaments: a spectroscopic study.

The principal aim of this investigation was to study the change of the protein flexibility and/or conformational properties of actin filaments upon the replacement of Ca2+ by Mg2+. The temperature dependence of the fluorescence lifetime and the anisotropy decay of N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) attached covalently to the Cys374 residue of actin were measured. Saturation transfer electron paramagnetic resonance (ST-EPR) experiments were also carried out using N-(1-oxyl-2,2,6, 6-tetramethyl-4-piperidinyl)-maleimide (MSL) attached to the same residue (Cys374). The Arrhenius analysis of the temperature dependence of the fluorescence lifetimes shows that for Mg-F-actin, both the activation energy (E*) and the frequency factor (A) are smaller than they are for Ca-F-actin. The longer rotational correlation times resolved in the fluorescence experiments are larger in the Mg2+-loaded form of the actin filament between 6 degreesC and 28 degreesC, but this difference becomes negligible above 28 degreesC. The results of saturation transfer electron paramagnetic resonance measurements on maleimide spin-labeled actin filaments indicate that the replacement of Ca2+ by Mg2+ induced a decrease of the mobility of the label on the sub-millisecond time scale. Based upon these results, we concluded that the filaments polymerized from Ca-actin are more flexible than the filaments of Mg-actin.

Actins↗
PubMed2001

Capability of arsonolipids to transport divalent cations: a Pressman cell study.

The ability of the newly synthesized arsonolipids (2,3-diacyloxyprophlarsonic acids) to transport cations was studied using the Pressman cell. Experimental results demonstrate that arsonolipids are much more efficient carriers of Ca(2+) and Mg(2+) than natural phosphatidic acid in the Pressman cell experiments. The ability of arsonolipids to transfer Ca(2+) is affected by the lipid side chain length in the order: C(12)>>C(14) approximately C(16). Ca(2+) is transferred faster than Mg(2+), suggesting that the latter is more tightly bound to the arsonolipids. The transfer kinetic curves are parabolic for C(12), while initially linear with a tendency to reach a steady state for C(14) and C(16), when the pH in the donor compartment was 8.3. The transport kinetics for both ions studied were best fitted by an equation derived from saturation kinetics that apply in reversible chemical reactions. The ion transfer rates increased as the pH in the donor compartment decreased.

Arsenicals↗
PubMed2002

Modulation of the human polycystin-L channel by voltage and divalent cations.

Polycystin-L (PCL) is highly homologous in sequence and membrane topology to polycystin-2, the product of the second gene responsible for autosomal dominant polycystic kidney disease (ADPKD). PCL and polycystin-2 were recently shown to be Ca2+-permeable, Ca2+-activated cation channels. Further characterization of polycystins will help in the understanding of cystogenesis and pathogenesis of ADPKD. In the present study, we expressed human PCL in Xenopus oocytes and studied its function utilizing patch-clamp and two-electrode voltage clamp techniques. In addition to its permeability to Ca2+, K+ and Na+, PCL was highly permeable to NH4+ and Cs+ with a permeability ratio NH4+:Cs+:Na+ of 2.2:1.02:1. Voltage modulation of channel properties was studied using cell-attached (C-A) and excised inside-out (I-O) patches. In the C-A mode, the open probability (NP(o)) of PCL at negative potentials (NP(o)=0.22) was higher than at positive potentials (NP(o)=0.05). The mean open time averaged 31.6 ms at negative potentials, and 6.2 ms at positive potentials; single-channel activity exhibited bursts with a mean interburst time of 178 ms. Using I-O patches under symmetrical ionic conditions, single-channel inward conductance was significantly larger than outward conductance, indicating a slight inward rectification. External Mg2+ inhibited the PCL channel currents. The inhibitory effect was voltage-dependent and substantially reduced by depolarization. The time course of inactivation depended on external calcium concentration but was independent of voltage and peak current. This study shows that although PCL is not a voltage-gated channel, its channel activity and inhibition by Mg2+ are modulated by membrane potential.

Animals↗