Divalent cation binding to ceruloplasmin

Binding of calcium to human and sheep ceruloplasniin was investigated by metal substitution with manganese and competitive displacement of bound manganese by calcium monitored by electron paramagnetic resonance spectroscopy. The K _d for calcium was found to be 1.4mm. Magnesium also bound to ceruloplasmin, with K _d = 0.3 and 0.7 mm for the human and sheep protein, respectively. The thermal stability of ceruloplasmin, as studied by differential scanning calorimetry, was affected by calcium but not by magnesium. A considerable increase of the T _m value, from 73.8 to 83.1°C, was observed for sheep ceruloplasmin in the presence of calcium. The T _m value of the human protein was only slightly altered by calcium (from 85.1 to 87°C). The interaction of ceruloplasmin with the chromatographic material used for its isolation, Sepharose 4B derivatized with chloroethylamine, was weakened by calcium. This allowed us to set up a novel purification scheme that made it possible to efficiently isolate ceruloplasmin and prothrombin from plasma with the same single-step chromatography.     

Divalent metal cation binding properties of human prothymosin alpha.

The divalent cation binding properties of human prothymosin alpha, an abundant nuclear protein involved in cell proliferation, were evaluated. By using prothymosin alpha retardation on a weak cation chelating resin charged with various divalent cations, specific binding of Zn2+ ions by prothymosin alpha was observed. This finding was further confirmed by the equilibrium dialysis analysis which demonstrated that, within the micromolar range of Zn2+ concentrations, prothymosin alpha could bind up to three zinc ions in the presence of 100 mM NaCl and up to 13 zinc ions in the absence of NaCl. Equilibrium dialysis analysis also revealed that prothymosin alpha could bind Ca2+, although the parameters of Ca2+ binding by prothymosin alpha were less pronounced than those of Zn2+ binding in terms of the number of metal ions bound, the KD values, and the resistance of the bound metal ions to 100 mM NaCl. The effects of Zn2+ and Ca2+ on the interaction of prothymosin alpha with its putative partners, Rev of HIV type 1 and histone H1, were examined. We demonstrated that Rev binds prothymosin alpha, and that prothymosin alpha binding to Rev but not to histone H1 was significantly enhanced in the presence of zinc and calcium ions. Our data suggest that the modes of prothymosin alpha interaction with Rev and histone H1 are distinct and that the observed zinc and calcium-binding properties of prothymosin alpha might be functionally relevant.     

Divalent cation binding to wheat germ calmodulin

The Ca2+ binding to plant (wheat germ) calmodulin was measured in 0.1 M NaCl by a flow-dialysis method. The four macroscopic binding constants best fitted to the data were 0.20, 0.25, 0.025, and 0.0024 microM-1. The cysteine residue of this calmodulin is located at the 27th position from the NH2-terminal (Yazawa, M. et al. (1982) Abstr. 33th Conf. Protein Structure pp. 9-12, Osaka). According to the quantitative analysis of the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) with Cys 27, the calmodulin which binds 3 Ca2+ showed the minimum reactivity with DTNB. This suggests that the site for the third Ca2+ binding is located close to Cys 27. Cys 27 was spin-labeled with N-(2,2,6,6-tetramethyl-4-piperidine-1-oxyl)maleimide, and its ESR spectrum was measured in the presence of Mn2+ and/or Ca2+. The rotational relaxation time of the label (1.2 ns) was increased by about one-tenth with 1 to 2 mol of bound Ca2+, but was unchanged with Mn2+. On the other hand, Mn2+ induced a remarkable quenching of the spectrum. From the decrease in the peak heights of the ESR spectrum, the distance between the label and the first bound Mn2+ was estimated to be 0.8 nm. It is concluded that the first Mn2+ binds to a domain near the NH2-terminal. The difference UV absorption spectrum induced by Mn2+ was similar to that induced by Ca2+. However, the amount of Mn2+ needed to saturate the difference spectrum was 1 mol more than the amount of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Divalent cation binding to the high- and low-affinity sites on G-actin

Metal binding to skeletal muscle G-actin has been assessed by equilibrium dialysis using 45Ca2+ and by kinetic measurements of the increase in the fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine-labeled actin. Two classes of cation binding sites were found on G-actin which could be separated on the basis of their Ca2+ affinity: a single high-affinity site with a Kd considerably less than 1 microM and three identical moderate-affinity binding sites with a Kd of 18 microM. The data for the Mg2+-induced fluorescence enhancement of actin labeled with N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine support a previously suggested mechanism [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886] in which Ca2+ is replaced by Mg2+ at the moderate affinity site(s), followed by a slow actin isomerization. This isomerization occurs independently of Ca2+ release from the high-affinity site. The fluorescence data do not support a mechanism in which this isomerization is directly related to Ca2+ release from the high-affinity site. Fluorescence changes of labeled actin associated with adding metal chelators are complex and do not reflect the same change induced by Mg2+ addition. Fluorescence changes in the labeled actin have also been observed for the addition of Cd2+ or Mn2+ instead of Mg2+. It is proposed actin may undergo a host of subtle conformational changes dependent on the divalent cation bound. We have also developed a method by which progress curves of a given reaction can be analyzed by nonlinear regression fitting of kinetic simulations to experimental reaction time courses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Divalent cation effects on membrane bending in heated erythrocytes

The thermal fragmentation of human erythrocytes involves either surface wave growth and membrane externalization at the cell rim or membrane internalization at the cell dimple. In symmetrical monovalent electrolytes an increase in membrane internalization at the cell dimple correlates with the decrease in zeta potential arising from surface charge (sialic acid residue) depletion. The influence of divalent cations on thermal fragmentation is examined in this work. The erythrocyte zeta potential decreased when divalent cations replaced some Na⁺ in the cell-suspending phase. The incidence of membrane internalization increased in rank order Ca²⁺>Ba²⁺>Mg²⁺Sr²⁺. Calcium continued to influence the thermal fragmentation of cells highly depleted of sialic acid, suggesting that the ion also interacted with membrane sites other than sialic acid. The divalent cation influence on cell fragmentation was shown to be greater than that due to zeta potential decrease alone. This conclusion was supported by the observation that the divalent cation-induced changes in zeta potential showed much less cation specificity than did the changes induced in the thermal fragmentation pattern. The result implies that the specificity of the divalent cation effects was due to interactions within the erythrocyte shear layer. The possibility that the interaction is with membrane lipids is examined.     

Divalent cation binding to reduced and octanoyl acyl-carrier protein

Mn(II) EPR binding studies with reduced acyl-carrier protein (ACP-SH) strongly suggest the presence of two relatively high-affinity manganese-binding sites (average Kd/site approximately 80 microM) at physiological pH. Lowering the pH or titrating with sodium chloride reduces the average number of bound divalent cations and decreases the binding affinity. This is consistent with the idea that anionic ligand(s), e.g. the carboxylate of glutamic or aspartic acid, on the protein are involved in manganese ion coordination. At pH values above 8.0, binding affinity is also reduced, whereas the average number of bound metal ions increases to about five at pH 8.5. By interacting weakly with divalent cations (average Kd/site approximately 1 mM), octanoyl acyl-carrier protein (OcoACP) exhibits dramatically different metal-ion-binding properties compared to ACP-SH. Calcium and magnesium can compete in either ACP species for manganese binding. Photochemically-induced dynamic nuclear polarisation 1H-NMR experiments strongly suggest that ACP-SH and OcoACP undergo at pH-induced conformational change between pH 5.5 and pH 7.0, and that divalent cations stabilize the protein against such pH-induced structural perturbations.     

Divalent cation binding to phospholipids: An EPR study

Summary Divalent cation association to sonicated phospholipid liposomes has been examined with electron paramagnetic spectroscopy. Spectra were obtained suggesting that, in some cases, divalent cations associated with acidic phospholipid head groups are highly mobile.Using the amplitude of its characteristic sextet signal as a measure of free Mn(H₂O) ₆ ⁺⁺ , the apparent affinities of cardiolipin and phosphatidylserine for Mn²⁺ were measured as a function of monovalent electrolyte. Monovalent cations having smaller nonhydrated radii were more effective in displacing Mn from the phospholipids. Under conditions of low divalent cation concentrations, it is shown that the Gouy-Chapman diffuse double layer theory predicts a Mn-affinity (K _A ) inversely proportional to the square of monovalent salt concentration. Although this relationship was closely obeyed for Mn binding to cardiolipin, the fall-off inK _A with added sodium chloride was slower in the cases of Mn binding to phosphatidylserine or phosphatidic acid.When phosphatidylcholine or cholesterol was incorporated into mixed vesicles along with a fixed amount of charged phospholipid, the Mn-binding strength was roughly proportional to the weight fraction of the latter. This result is consistent with: (1) a random dispersal of lipids in the bilayer, and (2) a 1:2 divalent cation-phospholipid interaction.     

Divalent cation effects on acetylcholine-activated channels at the frog neuromuscular junction

The effects of the divalent cations Ca and Mg on the properties of ACh-activated channels at the frog neuromuscular junction were studied using a two-microelectrode voltage clamp. The divalent cation concentration was varied from 2 to 40 mM in solutions containing 50% normal Na. The reversal potential was determined by interpolation of the acetylcholine (ACh)-induced current versus voltage relationship. The single-channel conductance and the mean channel lifetime were calculated from fluctuation analysis of the ACh-induced end-plate current. Extracellular Na and/or divalent cations affected the reversal potential of endplate channels in a way that cannot be described by the Goldman-Hodgkin-Katz equation or by a simple two-barrier, one-binding site model of the channel if the assumption was made that permeability ratios were constant and not a function of ion concentrations. Increasing the divalent cation concentration decreased the single-channel conductance to approximately 10 pS in solutions with 50% Na and 40 mM divalent cation concentrations. The effect of the divalent cations Ca and Mg on the mean channel lifetime was complex and dependent on whether the divalent cation was Ca or Mg. The mean channel lifetime was not significantly changed in most solutions with increased Ca concentration, while it was slightly prolonged by increased Mg concentration.     

Divalent cation binding to phospholipid vesicles. Dependence on temperature and lipid fluidity

Mn²⁺ binding to vesicles prepared from several different species of anionic phospholipids was determined as a function of temperature by electron paramagnetic resonance (EPR). The Mn²⁺ affinities of phosphatidylserine, cardiolipin and egg yolk phosphatidylglycerol all increased monitonically with temperature.Vesicles prepared from hydrogenated and natural (bovine) phosphatidylserine were monitored with respect to hydrocarbon chain fluidity as well as Mn²⁺ binding. Contrary to expectations based on surface potential considerations, the affinity of phosphatidylserine for divalent cations was apparently not lowered in going from the gel state to the liquid crystalline state of the bilayer. The results are instead consistent with an enhancement in cation affinity with increased lipid fluidity.Dipalmitoyl phosphatidylglycerol vesicle fluidity and Mn²⁺ binding were also studied with EPR. A large reduction in the measured Mn²⁺ affinity accompanied melting of the phospholipid, but observed hysteresis in the temperature dependence of the binding render uncertain any simple explanation based on changes in surface potential. Supplementary light scattering data indicated that vesicle aggregation was involved in the hysteresis phenomena.     

Divalent cation effects on calcineurin phosphatase: differential involvement of hydrophobic and metal binding domains in the regulation of the enzyme activity

Summary The effects of divalent metals, metal chelators (EDTA, EGTA) and sodium dodecyl sulfate were investigated on the phosphatase activity of isolated bovine brain calcineurin assayed in the absence (called intrinsic) and presence of calmodulin. Intrinsic phosphatase was increased by Mn²⁺, was unaffected by Mg²⁺, Ca^2–, and Ba⁺, and was markedly inhibited by Ni^2–, Fe²⁺, Zn²⁺ and Cu^2–. When assayed in the presence of calmodulin, many divalent metals (Ni^2–, Zn²⁺, Pb²⁺, Cd²⁺), besides Mn²⁺, increased modestly the phosphatase activity at low concentrations (10–100 M) and inhibited it markedly at high concentrations. Ca^2–-calmodulin stimulated phosphatase activity was antagonized by Ni²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Pb²⁺, at low concentrations (50 M), and by Ba²⁺, Cd²⁺ at slightly higher concentrations (> 100 M); Mn²⁺ and Co^2– (50 M to 1 mM) in fact augmented it. EDTA and EGTA in a concentration and time dependent fashion inhibited the intrinsic phosphatase activity, particularly that of trypsinized calcineurin. SDS in low concentrations (0.005%) augmented the phosphatase activity and inhibited it at high concentrations. Mn²⁺ (± calmodulin) and Ca²⁺ only with calmodulin present increased the phosphatase activity assayed with low concentrations of SDS. The EDTA dependent inhibition of intrinsic phosphatase was almost abolished in assays containing SDS. Prior exposure of calcineurin to Mn²⁺ led to a high activity conformation state of calcineurin that was long-lived or pseudo-irreversible. Such Mn²⁺-activated state of calcineurin exhibited no discerbible change in the affinity towards myelin basic protein or its inhibition by trifluoperazine. At alkaline pH, Mg²⁺ supported the intrinsic phosphatase activity, although to a lesser degree than Mn²⁺. The latter cation, compared to Mg²⁺ and Ni²⁺, was also a more powerful stimulator of the calcineurin phosphatase assayed with histone (III-S) and myosin light chain as substrates.     

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

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, we found that vinblastine and 1 mM Mn2+ or Ca2+ causes immediate condensation of tubulin. The predominant aggregates observed by electron microscopy are large sheets. This effect was not found with 1 mM Mg2+. At 100 microM cation concentrations (Mn2+, Mg2+, or Ca2+), GDP enhances vinblastine-induced spiral formation by 0.55 (+/- 0.26) kcal/mol. This effect is found only in K2, the association of liganded heterodimers at the ends of growing spirals. There is no GDP enhancement of K1, the binding of drug to heterodimer, although K1 is dependent upon the divalent cation concentration. NaCl diminishes tubulin condensation, probably by inhibiting lateral association, and allows an investigation of higher divalent cation concentrations. In the presence of 150 mM NaCl plus 1 mM divalent cations (Mn2+, Mg2+, or Ca2+) GDP enhances vinblastine-induced spiral formation by 0.35 (+/- 0.21) kcal/mol. Relaxation times determined by stopped-flow light scattering experiments in the presence of 150 mM NaCl and vincristine are severalfold longer than those in the presence of vinblastine, consistent with a mechanism involving the redistribution of longer polymers. Unlike previous results in the absence of NaCl, relaxation times in the presence of NaCl are only weekly protein concentration dependent, suggesting the absence of annealing or an additional rate-limiting step in the mechanism.     

Divalent cation binding to phospholipid veiscles. Dependence on temperature and lipid fluidity.

Mn2+ binding to vesicles prepared from several different species of anionic phospholipids was determined as a function of temperature by electron paramagnetic resonance (EPR). The Mn2+ affinities of phosphatidylserine, cardiolipin and egg yolk phosphatidylglycerol all increased monitonically with temperature. Vesicles prepared from hydrogenated and natural (bovine) phosphatidylserine were monitored with respect to hydrocarbon chain fluidity as well as Mn2+ binding. Contrary to expectations based on surface potential considerations, the affinity of phosphatidylserine for divalent cations was apparently not lowered in going from the gel state to the lipid crystalline state of the bilayer. The results are instead consistent with an enhancement in cation affinity with increased lipid fluidity. Dipalmitoyl phosphatidylglycerol vesicle fluidity and Mn2+ binding were also studied with EPR. A large reduction in the measured Mn2+ affinity accompained melting of the phospholipid, but observed hysteresis in the temperature dependence of the binding render uncertain any simple explanation based on changes in surface potential. Supplementary light scattering data indicated that vesicle aggregation was involved in the hysteresis phenomena.     

Divalent cation and hydrogen ion effects on the structure and surface activity of pulmonary surfactant

The structure and surface activity of the extracellular fraction of pulmonary surfactant known as tubular myelin are Ca2+ dependent. Previous studies have demonstrated surfactant-specific proteins with monomeric molecular weights of 28,000-36,000 (SP28-36) are associated with this fraction. In reassembled lipoprotein mixtures, SP28-36 promotes the Ca2+-induced aggregation and surface activity of surfactant lipids, but the detailed interactions between Ca2+, SP28-36, and surfactant lipids have not been established. In this study, we investigated the effect of various cations on the aggregation of surfactant lipid liposomes in the presence of SP28-36. SP28-36 reduced the threshold ion concentration for liposome aggregation from greater than 10 to 0.5 mM for Ca2+, Ba2+, and Sr2+ but not Mg2+ or Mn2+. The liposome aggregation was reversed by ethylenediaminetetraacetic acid and not associated with leakage of carboxyfluorescein. SP28-36 promoted similar liposome aggregation at pH less than 5 in the absence of divalent cations. Surfactant lipids adsorbed slowly to an air-fluid interface in all ionic conditions unless SP28-36 was present. Both Ca2+ and H+ induced rapid lipid adsorption in the presence of SP28-36. The surface activity of native surfactant had a similar ion dependence. Electron micrographs of native surfactant showed typical tubular myelin structures at pH 7.4 only in the presence of Ca2+. At pH 4.4 in the absence of Ca2+, similar but not identical structures were seen. In the reconstituted system, SP28-36 in the presence of Ca2+ induced the formation of larger multilayered structures including parallel bilayers and small areas of squares and triangles with dimensions similar to structures found in the native material.(ABSTRACT TRUNCATED AT 250 WORDS)     

Divalent cation sensitivity of BK channel activation supports the existence of three distinct binding sites

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 alpha subunit. Two mechanisms account for physiological regulation of BK channels by microM 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+, Sr2+, and Cd2+. Mn2+, Co2+, and Ni2+ produce little observable effect through the high affinity regulatory mechanisms, while all six divalent cations enhance activation through the low affinity mechanism defined by residue E399. Furthermore, each type of mutation affects kinetic properties of BK channels in distinct ways. The Ca2+ bowl mainly accelerates activation of BK channels at low [Ca2+], while the D362/D367-related high affinity site influences both activation and deactivation over the range of 10-300 microM Ca2+. The major kinetic effect of the E399-related low affinity mechanism is to slow deactivation at mM Mg2+ or Ca2+. The results support the view that three distinct divalent-cation binding sites mediate regulation of BK channels.     

Divalent cation binding specificities and microsphere formation of pyran copolymer and related polycarboxylates.

Pyran copolymer is a polyanionic drug with a broad spectrum of biologic activities. The present study focuses on the physicochemical interactions of pyran with divalent cations. Potentiometric titration curves of pyran are typical of the general polyelectrolyte behavior of high charge density polycarboxylates and show no apparent conformational transitions. The interaction of pyran with divalent cations follows an order of affinity for site binding, Mn²⁺ > Ca²⁺ > Mg²⁺, that is common also to four other polycarboxylates tested. The titration data indicate the formation of a soluble chelate complex, for which pyran, of the five polymers, shows the greatest propensity. Specific conditions in terms of pH and degree of neutralization are found under which complex formation is preceded by the precipitation of microspheres. However, only pyran and polyitaconic acid precipitate under conditions which are closest to physiologic. The experimental results suggest a mechanism for microsphere formation that is consistent with a hypothesized mechanism of pharmacologie action of pyran-divalent cation complexes.     

Divalent cation and lipid-protein interactions of biomembranes

Divalent cations play an important role in the functions of biomembranes. This review deals with three topics: (1) Mg²⁺-mediated change in physical state of phospholipid induces conformation and activity change of reconstituted mitochondrial H⁺-ATPase, (2) a proper transmembrane Ca²⁺ gradient is essential for the higher enzymatic activity of adenylate cyclase, and (3) role of transmembrane Ca²⁺ gradient in the modulation of reconstituted sarcoplasmic reticulm Ca²⁺-ATPase activity.     

Divalent cation transport systems of Rhodopseudomonas capsulata.

Separate divalent cation transport systems for energy-dependent uptake of Mg2+ and Mn2+ were found both with aerobically and heterotrophically grown and with photosynthetically grown cells of Rhodopseudomonas capsulata. The maximum rate of Mg2+ uptake differed between photosynthetic and aerobic cells, while the Km for the Mg2+ transport system was constant. Photosynthetic midlog-phase cells exhibited Km's for uptake of about 55 micrometer Mg2+ and 0.5 micrometer Mn2+. The Vmax's also differed between the two systems: 0.6 to 1.8 mumol/min per g (dry weight) of cells for Mg2+, but only 0.020 mumol/min per g for Mn2+, making the distinction between a "macro-requirement" system and a system functioning at trace nutrient levels. Calcium was not normally taken up by intact cells of R. capsulata. However, chromatophore membranes isolated from photosynthetic cells took up Ca2+ by an energy-dependent process.