Thermodynamics of binding of calcium, magnesium, and zinc to the N-methyl-D-aspartate receptor ion channel peptidic inhibitors, conantokin-G and conantokin-T

The binding isotherms of the divalent metal cations, Ca2+, Mg2+, and Zn2+, to the synthetic gamma-carboxyglutamic acid-containing neuroactive peptides, conantokin-G (con-G) and conantokin-T (con-T), have been determined by isothermal titration calorimetry (ITC) at 25 degreesC and pH 6.5. We have previously shown by potentiometric measurements that con-G contains 2-3 equivalent Ca2+ sites with an average Kd value of 2800 microM. With Mg2+ as the ligand, two separate exothermic sites are obtained by ITC, one of Kd = 46 microM and another of Kd = 311 microM. Much tighter binding of Zn2+ is observed for these latter two sites (Kd values = 0.2 microM and 1.1 microM), and a third considerably weaker binding site is observed, characterized by a Kd value of 286 microM and an endothermic enthalpy of binding. con-T possesses a single exothermic tight binding site for Ca2+, Mg2+, and Zn2+, with Kd values of 428 microM, 10.2 microM, and 0.5 microM, respectively. Again, in the case of con-T, a weak (Kd = 410 microM) endothermic binding site is observed for Zn2+. The binding of these cations to con-G and con-T result in an increase in the alpha-helical content of the peptides. However, this helix is somewhat destabilized in both cases by binding of Zn2+ to its weakest site. Since the differences observed in binding affinities of these three cations to the peptides are substantially greater than their comparative Kd values to malonate, we conclude that the structure of the peptide and, most likely, the steric and geometric properties imposed on the cation site as a result of peptide folding greatly influence the strength of the interaction of cations with con-G and con-T. Further, since the Zn2+ concentrations released in the synaptic cleft during excitatory synaptic activity are sufficiently high relative to the Kd of Zn2+ for con-G and con-T, this cation along with Mg2+, are most likely the most significant metal ion ligands of these peptides in neuronal cells.     

Molecular regulation of the interaction between leukocyte function-associated antigen-1 and soluble ICAM-1 by divalent metal cations

Surface plasmon resonance (SPR) was used to investigate and characterize the interaction between LFA-1 and sICAM-1 (a soluble form of ICAM-1). Full-length LFA-1 was immobilized on a hydrophobic surface, and sICAM-1 binding was monitored in a flow cell format. The binding of sICAM-1 to LFA-1 was specific and dependent upon Mg2+; Abs to both sICAM-1 and LFA-1 blocked the interaction, and EDTA abolished all binding. Association and dissociation rate constants (k(a) and k(d), respectively) for sICAM-1 were 2.24 x 10(5) M(-1) s(-1) and 2.98 x 10(-2) s(-1), respectively, giving a calculated K(ICAM) of 133 nM. Since the LFA-1/ICAM-1 interaction is highly sensitive to the presence of metal cations, SPR was also used to probe the affinity of the metal binding sites. The K(Mg) values were 160 and 12 microM in the absence (EGTA) and the presence of Ca2+ (100 microM), respectively; in addition, K(Mn) was 2 microM in the presence of Ca2+ (100 microM). Increasing Ca2+ into the millimolar concentration range, however, resulted in a competitive displacement of Mg2+/Mn2+ and decreased sICAM-1 binding. Based on these data, a synergistic model for the molecular regulation of LFA-1 by divalent metal cations is proposed, and implications to cellular adhesion are discussed.     

Protective effect of dehydroepiandrosterone against copper-induced lipid peroxidation in the rat

This study investigates the effectiveness and multitargeted activity of dehydroepiandrosterone (DHEA) as antioxidant in vivo. A single dose of DHEA was given IP to male rats. Liver and brain microsomes, and plasma low density lipoprotein (LDL), were isolated from rats sacrificed 17 h later. Liver and brain microsomes were challenged with CuSO(4) and, as index of lipid peroxidation, the production of thiobarbituric acid reactive substances (TBARS) was measaured. Also, plasma low-density lipoprotein (LDL) were challenged with copper and the time course of lipid peroxidation was evaluated following the formation of conjugated dienes. The onset of TBARS generation induced by copper was marked delayed in both liver and brain microsomes from DHEA-treated animals. Also, the resistance of LDL to oxidation, expressed by the duration of the lag-phase of the kinetic curve, was significantly enhanced in DHEA-treated rats. Results indicate that in vivo DHEA supplementation makes subcellular fractions isolated from different tissues and plasma constituents (LDL) more resistant to lipid peroxidation triggered by copper. The antioxidant effect on plasma LDL might be of special relevance to the proposed antiatherogenic activity of DHEA. Moreover, multitargeted antioxidant activity of DHEA might protect tissues from oxygen radicals damage.     

Different mechanisms are progressively recruited to promote Cu(II) reduction by isolated human low-density lipoprotein undergoing oxidation

The kinetics of Cu(II) reduction and its relationship to the process of low density lipoprotein (LDL) oxidation were investigated in isolated human LDL incubated with CuSO4 by using the Cu(I) chelator and indicator dye bathocuproine disulfonate (BC). The inclusion of BC in the incubation medium containing isolated LDL and different concentrations of CuSO4 revealed a biphasic kinetics of Cu(II) reduction consisting of an early phase followed by a plateau phase and a subsequent extensive reduction phase. The amount of Cu(I) formed during the early phase, as well as the rate of its generation, were strictly dependent on both the level of Cu(II) available (saturation was observed at 20 and 50 microM CuSO4) and the concentration of alpha-tocopherol within native LDL particles. Artificial enrichment of LDL with different concentrations of alpha-tocopherol led to a parallel increase of both the amount of Cu(II) reduced and the rate of reduction. The late phase of Cu(II) reduction was strictly related to the availability of copper but was largely independent from alpha-tocopherol. Neither the amount of Cu(I) generated nor the rate of generation were saturated at concentrations of copper up to 100 microM. Comparable results were obtained by adding BC at different time-points to the LDL-copper mixture, in order to measure at the same time-points both the true rate of Cu(II) reduction and the generation of TBARS during the dynamic process of LDL oxidation. The rate of Cu(II) reduction was already high during the lag-phase of the LDL oxidation profile and progressively decreased as alpha-tocopherol concentration decreased. The subsequent increase in the rate of Cu(II) reduction paralleled the formation of TBARS during the extensive LDL oxidation phase. These results suggest that different mechanisms of Cu(II) reduction, namely alpha-tocopherol-dependent and independent (likely lipid peroxide-dependent), are progressively recruited during copper-promoted LDL oxidation.     

Effects of divalent cations, protons and calmidazolium at the rat P2X7 receptor

The P2X7 receptor is a uniquely bifunctional molecule through which ATP can open a small cationic channel typical of ionotropic receptors and also induce a large pore permeable to high molecular weight molecules (> 600 Da). Activation of this large pore can lead to cell lysis within 1-2 min. We asked whether pharmacological differences existed between the cationic channel and the cell permeabilizing pore by measuring whole-cell currents and uptake of a propidium dye (YO-PRO; Mw 629) in HEK293 cells stably expressing the rat P2X7 receptor, and comparing the actions of divalent cations and protons in these two assays. Currents in response to 2'-3'-(O)-(4-benzoyl benzoyl) ATP (BzATP, 30 microM) were inhibited by extracellular calcium, magnesium, zinc, copper and protons with half-maximal inhibitory concentrations (IC50) of 2.9 mM, 0.5 mM, 11 microM, 0.5 microM and 0.4 microM, respectively. The inhibition was voltage independent in each case. YO-PRO uptake induced by BzATP was also inhibited with similar IC50 values. The rank order of potency of a range of divalents was Cu2+ > Cd2+ = Zn2+ > Ni2+ > Mg2+ = Co2+ > Mn2+ > Ca2+ = Ba2+ > Sr2+. These results suggest that these divalent cations and protons all act primarily as allosteric modulators to alter the affinity of ATP binding to the P2X7 receptor. In contrast, extracellular (but not intracellular) calmidazolium inhibited the BzATP-evoked current by up to 90% (IC50 = 15 nM) but had no effect on YO-PRO uptake. Thus, calmidazolium can block activation of the ionic channel but this does not prevent the formation of the large permeabilizing pore.     

Assessing food choice in school children: reliability and construct validity of a method stacking food photographs

Cigarette smoke of which the major component is nicotine plays an important role in the development of cardiovascular diseases. To study the effect of in vitro incubation of LDL with nicotine and its metabolite, cotinine on a copper-induced peroxidation, we monitored the formation of conjugated dienes, hydroperoxides and thiobarbituric acid-reactive substances production. The LDL studied were taken from six non-smokers (aged 41.5 years) and six smokers who consumed at least ten cigarettes per day (40.7 years). LDL oxidation with CuSO4 showed that cigarette smoking promotes LDL susceptibility to peroxidative modification. During the peroxidation of LDL with nicotine (O to 5 mmol/1) and CuSO4 (5 micromol/l), the formation of hydroperoxides decreased when nicotine concentrations increased and the production of TBARS increased in a concomitant manner. The results showed that the presence of nicotine destabilized the production of hydroperoxides in LDL and increased the formation of secondary oxidation products. On the other hand, cotinine had no effect on LDL oxidative susceptibility in smokers and non-smokers.     

Tattoos on women: marks of distinction or abomination?

The regulatory properties of the divalent metal ions Mg2+, Ca2+ and Mn2+ on the activity and kinetic behaviour of rat liver microsomal cholesterol esterase were studied in vitro. Mg2+ and Ca2+ exhibited similar concentration and preincubation time-dependent increases in esterase activity, with maximal stimulation at a concentration of 2 mM. However, Mn2+ had no effect at this concentration but displayed a potent inhibitory effect at concentrations above 20 mM. Activation of cholesterol esterase by Mg2+ and Ca2+ was selective in relation to i) the changes that cations produced in the enzyme kinetic constants, and ii) the chelating agents that reversed the metal ion-induced activation. Hence, the maximum rate of cholesterol ester hydrolysis doubled in the presence of Mg2+ and activation was reversed by EDTA, whereas a significant decrease in the apparent Km for cholesterol oleate was found when Ca2+ was added and this effect was blocked by ATP and EGTA. Both cations were able to reactivate cholesterol ester hydrolase activity in metal-depleted microsomes.     

Binding of different divalent cations to the active site of avian sarcoma virus integrase and their effects on enzymatic activity

Retroviral integrases (INs) contain two known metal binding domains. The N-terminal domain includes a zinc finger motif and has been shown to bind Zn2+, whereas the central catalytic core domain includes a triad of acidic amino acids that bind Mn2+ or Mg2+, the metal cofactors required for enzymatic activity. The integration reaction occurs in two distinct steps; the first is a specific endonucleolytic cleavage step called "processing," and the second is a polynucleotide transfer or "joining" step. Our previous results showed that the metal preference for in vitro activity of avian sarcoma virus IN is Mn2+ > Mg2+ and that a single cation of either metal is coordinated by two of the three critical active site residues (Asp-64 and Asp-121) in crystals of the isolated catalytic domain. Here, we report that Ca2+, Zn2+, and Cd2+ can also bind in the active site of the catalytic domain. Furthermore, two zinc and cadmium cations are bound at the active site, with all three residues of the active site triad (Asp-64, Asp-121, and Glu-157) contributing to their coordination. These results are consistent with a two-metal mechanism for catalysis by retroviral integrases. We also show that Zn2+ can serve as a cofactor for the endonucleolytic reactions catalyzed by either the full-length protein, a derivative lacking the N-terminal domain, or the isolated catalytic domain of avian sarcoma virus IN. However, polynucleotidyl transferase activities are severely impaired or undetectable in the presence of Zn2+. Thus, although the processing and joining steps of integrase employ a similar mechanism and the same active site triad, they can be clearly distinguished by their metal preferences.     

Modulation of GABAC response by Ca2+ and other divalent cations in horizontal cells of the catfish retina

GABAC responses were recorded in cultured cone-driven horizontal cells from the catfish retina using the patch clamp technique. At a holding potential of -49 mV, a bicuculline-resistant inward current (IGABA) was observed when 10 microM GABA was applied. The amplitude of IGABA increased as the extracellular Ca2+ ([Ca2+]o) was increased. Concentration-response curves of IGABA at 2.5 and 10 mM -Ca2+-o had similar EC50 (3.0 and 3.1 microM) and Hill coefficients (1.54 and 1. 24). However, the maximal response estimated at 10 mM [Ca2+]o was larger than the maximal response at 2.5 mM [Ca2+]o. Increasing Ca influx through voltage-gated Ca channels and the resulting rise in the intracellular Ca2+ concentration had no effects on IGABA. However, IGABA was inhibited by extracellular divalent cations, with the following order of the inhibitory potency: Zn2+ > Ni2+ > Cd2+ > Co2+. The inhibitory action of Zn2+ on the [Ca2+]o-dependent IGABA increase was noncompetitive. The action of [Ca2+]o on IGABA was mimicked by Ba2+ or Sr2+. These results demonstrate that the extracellular domain of GABAC receptors has two functionally distinct binding sites represented by Ca2+ (facilitation) and Zn2+ (inhibition). Since [Ca2+]o and [Zn2+]o change into the opposite direction by light, it seems likely that they modify cooperatively the efficacy of the positive feedback consisting of the GABAC receptor.     

Kinetic uniformity of the ATP hydrolysis reaction catalyzed by Mg2+-ATPase in smooth muscle cell membrane

The kinetic properties of Mg(2+)-ATPase (EC 3.6.1.3) from myometrium cell plasma membranes have been studied. Under conditions of enzyme saturation with ATP (0.5-1.0 mM) or Mg2+ (1.0-5.0 mM) the initial maximal rates of the Mg(2+)-dependent enzymatic ATP hydrolysis, V0 ATP and V0 Mg, are 27.4 +/- 3.3 and 25.2 +/- 4.1 mumol Pi/hour/mg of protein, respectively. The apparent Michaelis constant, Km, for ATP and of the apparent activation constant, K alpha, for Mg2+ are equal to 28.1 +/- 2.6 and 107.0 +/- 26.0 microM, respectively. The bivalent metal ions used at 1.0 mM suppress the Mg(2+)-dependent hydrolysis of ATP whose efficiency decreases in the following order: Cu2+ > Zn2+ = Ni2+ > Mn2+ > Ca2+ > Co2+. Alkalinization of the incubation medium from pH 6.0 to pH 8.0 stimulates the Mg(2+)-dependent hydrolysis of ATP. It has been found that Mg(2+)-ATPase has the properties of an H(+)-sensitive enzymatic sensor which is characterized by a linear dependence between the initial maximal rate of the reaction, V0, and the pH value. The feasible role of plasma membrane Mg(2+)-ATPase in some reactions responsible for the control of proton and Ca2+ homeostasis in myometrium cells has been investigated.     

An evaluation of the differences in human evoked cortical potentials to visual stimuli

Saccharomyces cerevisiae nuclei possess a polyphosphatase activity which is insensitive to a number of inhibitors of ATPase and pyrophosphatase (PPase) activities of the same organelle. Heparin, an effective inhibitor of the nuclear polyphosphatase activity, does not alter either the ATPase and PPase activity. The nuclear polyphosphatase activity is optimal at pH 7.5. Bivalent metal cations stimulate this activity in the following order: Co2+ > Mg2+ > Zn2+ > Mn2+. However, the magnitude of the stimulating effect is much lower than that for the polyphosphatase activities from other organelles of the same yeast. The polyphosphatase activity is nearly the same for polyphosphates ranging from [symbol: see text] = 9 to [symbol: see text] = 208, but is 1.5 times higher for tripolyphosphate. The K(m) values for the hydrolysis of polyphosphates with chain lengths [symbol: see text] = 3, 15 and 208 are 100, 5 and 4.1 microM, respectively. The polyphosphatase activity differs in some properties from that of the cell envelope, cytosol and vacuoles of the same S. cerevisiae strain.     

Antioxidant properties of butein isolated from Dalbergia odorifera

The antioxidant properties of butein, isolated from Dalbergia odorifera T. Chen, were investigated in this study. Butein inhibited iron-induced lipid peroxidation in rat brain homogenate in a concentration-dependent manner with an IC50, 3.3+/-0.4 microM. It was as potent as alpha-tocopherol in reducing the stable free radical diphenyl-2-picrylhydrazyl (DPPH) with an IC0.200, 9.2+/-1.8 microM. It also inhibited the activity of xanthine oxidase with an IC50, 5.9+/-0.3 microM. Besides, butein scavenged the peroxyl radical derived from 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) in aqueous phase, but not that from 2,2-azobis(2, 4-dimethylvaleronitrile) (AMVN) in hexane. Furthermore, butein inhibited copper-catalyzed oxidation of human low-density lipoprotein (LDL), as measured by conjugated dienes and thiobarbituric acid-reactive substance (TBARS) formations, and electrophoretic mobility in a concentration-dependent manner. Spectral analysis revealed that butein was a chelator of ferrous and copper ions. It is proposed that butein serves as a powerful antioxidant against lipid and LDL peroxidation by its versatile free radical scavenging actions and metal ion chelation.     

Inhibition studies of the carnitine acetyltransferase from skeletal muscle of the camel (Camelus dromedarius) by sulfhydryl reagents and metal ions

The effect of certain sulfhydryl reagents and metal ions were studied on the carnitine acetyltransferase (CAT) activity from the skeletal muscle of the Arabian camel (Camelus dromedarius). DTNB and iodoacetamide caused concentration and time dependent inhibition of CAT activity. The inhibition seen with these sulfhydryl reagents could be protected with prior incubation of the enzyme with acetyl-Co A, suggesting that these reagents might interact with the same site. Among the various metal ions tested, Cu2+, Zn2+ and Hg2+ caused total inhibition at very low concentrations, while, Mn2+, Mo6+ and Co2+ caused between 32-52% inhibition at 10 mM concentrations. Alkali earth divalent metals Mg2+ and Ca2+ caused less than 15% inhibition at this concentration. These metal ions are probably interacting at certain nucleophilic groups in the enzyme thus disrupting its tertiary structure.     

Calcium ion binding to thrombospondin 1

We have quantified the binding of Ca2+ to platelet thrombospondin 1 (TSP1) using equilibrium dialysis with 45CaCl2. Ca2+ binding to TSP1 was found to be cooperative with 10% occupancy at 15-20 microM CaCl2, 90% occupancy at 100 microM CaCl2, and a Hill coefficient of 2.4 +/- 0.2 The average apparent Kd was 52 +/- 5 microM. Maximum binding, assuming Mr = 450,000 and epsilon = 0.918 (A280/mg/ml), was 35 +/- 3 Ca2+/TSP1. This value is close to the 33 sites (11 per subunit) predicted based on homology of the epidermal growth factor (1 site) and aspartate-rich (10 sites) regions to known Ca2+ binding sequences. Ca2+ protected the aspartate-rich region from trypsin proteolysis, but not until nearly all of the Ca2+ binding sites were filled. At lower occupancy of Ca2+ binding sites, several limited tryptic digest products were obtained. This finding and the previous demonstration of extensive thiol-disulfide isomerization within the aspartate-rich regions suggest that subregions of the aspartate-rich region are stabilized in different conformers. Zn2+, Cu2+, Mn2+, Mg2+, Co2+, Cd2+, and Ba2+ were tested for their ability to modulate Ca2+ binding and protease sensitivity of TSP1. Zn2+ inhibited 40% of the Ca2+ binding but neither protected TSP1 from trypsin proteolysis, nor labilized TSP1 toward trypsin proteolysis. These results provide direct evidence for high capacity, cooperative and specific binding of Ca2+ to conformationally labile aspartate-rich repeats of TSP1.     

Dual control by divalent cations and mitogenic cytokines of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity expressed by human hemopoietic cells

Beta-1 integrins have essential functions in hemopoietic and immune systems by controlling phenomenons such as cell homing and cell activation. The function alpha 4 beta 1 and alpha 5 beta 1 integrins is regulated by divalent cations and, as demonstrated more recently, by mitogenic cytokines which activate them by "inside-out" mechanisms. Using the adhesive interaction of a cytokine-dependent human hemopoietic cell line to immobilized fibronectin, we have analyzed the requirements in divalent cations Mn2+, Mg2+ and Ca2+ for alpha 4 beta 1 and alpha 5 beta 1 activation by "inside-out" mechanisms triggered by cytokines such as granulocyte-macrophage colony stimulating factor or KIT ligand, or by external conformational constraints with the function-activating anti-beta 1 integrin monoclonal antibody 8A2. The intrinsic difference between these two modes of beta 1 integrin activation was revealed by their different requirements in divalent cations. We found that in the absence of any divalent cations, alpha 4 beta 1 and alpha 5 beta 1 were non-functional even after further stimulation by cytokines or 8A2. However, whilst either Ca2+, Mg2+ or Mn2+ were able to restore adhesive functions of alpha 4 beta 1 and alpha 5 beta 1 when activated by 8A2, only Mg2+ and Mn2+ were able to support activation of alpha 4 beta 1 and alpha 5 beta 1 by cytokines. Furthermore, high concentrations of Ca2+ exceeding 20 mM dramatically inhibited cell adhesion to fibronectin induced by Mn2+ and cytokines but not by 8A2. On the contrary, in the presence of both Ca2+ and Mg2+, Mn2+ had an additive effect on the activation of alpha 4 beta 1 and alpha 5 beta 1 by mitogenic cytokines. The presence of the absence of these divalent cations did not inhibit early tyrosine phosphorylation induced by the binding of KIT ligand to its tyrosine-kinase receptor KIT. Therefore, we propose that in hemopoietic cells, Ca2+, Mg2+ and Mn2+ may modulate in vivo alpha 4 beta 1 and alpha 5 beta 1 regulation by mitogenic cytokines, a phenomenon involved in the regulation of hemopoietic progenitor cell homing within the bone marrow.     

Inhibition by inorganic ions of a sustained calcium signal evoked by activation of mGlu5 receptors in rat cortical neurons and glia

The effect of mGlu receptor agonists on intracellular calcium (Ca2+) in rat cortical neurons and glial cells was studied. The responses evoked consisted of two phases; an initial transient response followed by a sustained plateau. In both cell types the order of potency of group I mGlu receptor agonists was DHPG > 1S,3R ACPD > 3-HPG. The selective mGlu5 agonist CHPG elicited responses in both cell types as did S4C3-HPG which is thought to be an mGlu5 agonist at high concentrations. S4-CPG had no effect on intracellular Ca2+ levels nor did it inhibit the action of IS,3R ACPD. These results suggest that the responses in both cell types are mediated by mGlu5 receptors. In the absence of extracellular Ca2+ ions, 1S,3R ACPD (100 microM) induced only a transient Ca2+ response which decayed to baseline with a time constant of approximately 20 s in both cell types. Subsequent readdition of Ca2+ (2 mM) to the external solution in the continued presence of 1S,3R ACPD induced a sustained Ca2+ plateau. The sustained Ca2+ plateau could be blocked by a number of inorganic cations, with an order of potency of Zn2+ > or = La3+ > Cd2+ > or = Co2+ > Ni2+ > Mg2+. Similar concentrations of Zn2+ had little effect on Ca2+-influx evoked by 25 mM K+. It is concluded that the Ca2+-entry pathway activated by mGlu5 receptors resembles store-operated Ca2+-entry pathways that have been described in other cell types.     

Oligomerization and divalent ion binding properties of the S100P protein: a Ca2+/Mg2+-switch model

S100P is a 95 amino acid residue protein which belongs to the S100 family of proteins containing two putative EF-hand Ca2+-binding motifs. In order to characterize conformational properties of S100P in the presence and absence of divalent cations (Ca2+, Mg2+ and Zn2+) in solution, we have analyzed hydrodynamic and spectroscopic characteristics of wild-type and several variants (Y18F, Y88F and C85S) of S100P using equilibrium centrifugation, gel-filtration chromatography, circular dichroism and fluorescence spectroscopies. Analysis of the experimental data shows the following. (1) In agreement with the predictions there are two Ca2+-binding sites in the S100P molecule with different affinity; the high affinity binding site has an apparent binding constant of approximately 10(7) M-1 and the low affinity binding site has an apparent binding constant of approximately 10(4) M-1. (2) The high and low affinity Ca2+-binding sites are located in the C and N-terminal parts of the S100P molecule, respectively. (3) These C and N-terminal sites can also bind other divalent ions. The C-terminal site binds Zn2+ (with relatively low affinity approximately 10(3) M-1), but not Mg2+. The N-terminal site binds Mg2+ with the apparent binding constant approximately 10(2) M-1. (4) Binding of Ca2+ to the C-terminal site and binding of Mg2+ to the N-terminal site occur in the physiological concentration range of these ions (micromolar for Ca2+ and millimolar for Mg2+). (5) Oligomerization state of the S100P molecule appears to change upon addition of Ca2+. On the basis of these observations a plausible model for S100P as a Ca2+/Mg2+ switch has been proposed.     

Smoking and female infertility: a systematic review and meta-analysis

The identification of Ca2+ as a cofactor in photosynthetic O2 evolution has encouraged research into the role of Ca2+ in photosystem II (PSII). Previous methods used to identify the number of binding sites and their affinities were not able to measure Ca2+ binding at thermodynamic equilibrium. We introduce the use of a Ca2(+)-selective electrode to study equilibrium binding of Ca2+ to PSII. The number and affinities of binding sites were determined via Scatchard analysis on a series of PSII membrane preparations progressively depleted of the extrinsic polypeptides and Mn. Untreated PSII membranes bound approximately 4 Ca2+ per PSII with high affinity (K = 1.8 microM) and a larger number of Ca2+ with lower affinity. The high-affinity sites are assigned to divalent cation-binding sites on the light-harvesting complex II that are involved in membrane stacking, and the lower-affinity sites are attributed to nonspecific surface-binding sites. These sites were also observed in all of the extrinsic polypeptide- and Mn-depleted preparations. Depletion of the extrinsic polypeptides and/or Mn exposed additional very high-affinity Ca2(+)-binding sites which were not in equilibrium with free Ca2+ in untreated PSII, owing to the diffusion barrier created by the extrinsic polypeptides. Ca2(+)-depleted PSII membranes lacking the 23 and 17 kDa extrinsic proteins bound an additional 2.5 Ca2+ per PSII with K = 0.15 microM. This number of very high-affinity Ca2(+)-binding sites agrees with the previous work of Cheniae and co-workers [Kalosaka, K., et al. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) pp 721-724, Kluwer, Dordrecht, The Netherlands] whose procedure for Ca2+ depletion was used. Further depletion of the 33 kDa extrinsic protein yielded a sample that bound only 0.7 very high-affinity Ca2+ per PSII with K = 0.19 microM. The loss of 2 very high-affinity Ca2(+)-binding sites upon depletion of the 33 kDa extrinsic protein could be due to a structural change of the O2-evolving complex which lost 2-3 of the 4 Mn ions in this sample. Finally, PSII membranes depleted of Mn and the 33, 23, and 17 kDa extrinsic proteins bound approximately 4 very high-affinity Ca2+ per PSII with K = 0.08 microM. These sites are assigned to Ca2+ binding to the vacant Mn sites.     

吸附材料处理含Zn2+重金属废水的影响因素

考察了废水初始pH值、不同阳离子、阴离子、有机物、共存重金属离子、极端条件等因素对吸附Zn2的影响.结果表明,8h后吸附接近饱和,废水初始pH =6时处理效果最好,Zn2+吸附容量为3.98 mg/g,去除率为79.6％;Na+、K+、Mg2+、Ca2+均会抑制Zn2的吸附,影响顺序从小到大为Na+＜K+＜ Mg2+＜ Ca2+;Cl-、SO2-4对吸附Zn2+抑制作用很小,且Cl-的抑制作用小于SO2-4;COD对吸附Zn2+有促进作用;Pb2+、Cu2+共存时,对Zn2+的吸附有抑制作用,影响顺序为Pb2+单元体系＜ Cu2+单元体系＜ Pb2+、Cu2+二元体系;高盐和强酸对Zn2+的吸附有较大影响,但高温基本无影响.饱和吸附材料的后处理试验表明,在550℃马弗炉中热处理6.5h,烧失率为75.4％,烧渣中Zn2+含量为1.75％,与热处理前相比,富集倍数为4.1倍.本文研究成果为吸附法处理含锌重金属废水并回收废水中的重金属提供了重要依据.     

Induced and synchronized estrus in cattle: dose titration of estradiol benzoate in peripubertal heifers and postpartum cows after treatment with an intravaginal progesterone-releasing insert and prostaglandin F2alpha

Seven cysteine-rich repeats form the ligand-binding region of the low-density lipoprotein (LDL) receptor. Each of these repeats is assumed to bind a calcium ion, which is needed for association of the receptor with its ligands, LDL and beta-VLDL. The effects of metal ions on the folding of the reduced N-terminal cysteine-rich repeat have been examined by using reverse-phase high-performance liquid chromatography to follow the formation of fully oxidized isomers with different disulfide connectivities. In the absence of calcium many of the 15 possible isomers formed on oxidation, whereas in its presence the predominant product at equilibrium had the native disulfide bond connectivities. Other metals were far less effective at directing disulfide bond formation: Mn2+ partly mimicked the action of Ca2+, but Ba2+, Sr2+, and Mg2+ had little effect. This metal-ion specificity was also observed in two-dimensional 1H NMR spectral studies; only Ca2+ induced the native three-dimensional fold. The two paramagnetic ions, Gd3+ and Mn2+, and Cd2+ did not promote adoption of a well-defined structure, and the two paramagnetic ions did not displace calcium ions. The location of calcium ion binding sites in the repeat was also explored by NMR spectroscopy. The absence of chemical shift changes for the side chain proton resonances of Asp26, Asp36, and Glu37 from pH 3.9 to 6.8 in the presence of calcium ions and their proximal location in the NMR structures implicated these side chains as calcium ligands. Deuterium exchange NMR experiments also revealed a network of hydrogen bonds that stabilizes the putative calcium-binding loop.