The activation of concanavalin A by lanthanide ions.

Divalent cadmium and lead and the trivalent lanthanides bind in the trasition metal site (S1) of concamavanlin A and induce saccharide binding to the protein in the presence of calcium. Partial activation of the protein in the presence of lanthanides alone indicates these ions bind into both transition metal (S1) and calcium sites (S2). The activity of a lanthanide-protein derivative may be increased by the addition of either calcium or a transition metal ion. The saccharide binding activity decreases in the order Zn2+ is greater than Ni2+ is greater than Co2+ is greater than Mn2+ is greater than Cd2+ reflecting the order of binding constants for these ions in the transition metal site. Like the lanthanides, divalent cadmium substitutes for both the transition metal ion and calcium ion to partially activate the protein. Divalent lead substitutes only for the transition metal ion and partially activates the protein upon addingcalcium. The data are consistent with a model in which saccharide binding activity is independent of the metal size in S1 but critically dependent upon metal size in S2.     

The influence of calcium ions on fibrin polymerization

The influence of calcium ions on the polymerization induced in fibrinogen solutions by thrombin and by Reptilase has been investigated by meansof static and dynamic light scattering in combination with measurements of the release of the fibrinopeptide A. The calcium concentration was varied in the range between 0.3 and 103 calcium ions per fibrinogen molecule. The enzyme concentration was chosen sufficiently low so that it was possible to make quantitative observations as a function of time, in particular, beforethe onset of gelation. Likewise, the influence of calcium ions on the enzymatically induced polymerization of fragment X was studied. The results indicate that there are at least three mechanisms by which calcium can influence the evolution of the polymer system on the path to gelation and clotting. Which mechanism dominates depends upon the calcium concentration.     

The role of calcium ions in fibrin polymerization

The article reviews the literature and authors' own data on the role of Ca ions in blood coagulation, namely, in the process of the formation of highly ordered fibrin clot. It has been shown that the main role of Ca2+ is the timely formation and stabilization of fibrin polymerization sites at all successive stages of the fibrin polymerization process.     

Structural characteristics of protein binding sites for calcium and lanthanide ions

Surveys of X-ray structures of Ca2+-containing and lanthanide ion-containing proteins and coordination complexes have been performed and structural features of the metal binding sites compared. A total of 515 structures of Ca2+-containing proteins were considered, although the final data set contained only 44 structures and 60 Ca2+ binding sites with a total of 323 ligands. Eighteen protein structures containing lanthanide ions were considered with a final data set containing eight structures and 11 metal binding sites. Structural features analysed include coordination numbers of the metal ions, the identity of their ligands, the denticity of carboxylate ligands, and the type of secondary structure from which the ligands are derived. Three general types of calcium binding site were identified in the final data set: class I sites supply the Ca2+ ligands from a continuous short sequence of amino acids; class II sites have one ligand supplied by a part of the amino acid sequence far removed from the main binding sequence; and class III sites are created by amino acids remote from one another in the sequence. The abundant EF-hand type of Ca2+ binding site was under-represented in the data set of structures analysed as far as its biological distribution is concerned, but was adequately represented for the chemical survey undertaken. A turn or loop structure was found to provide the bulk of the ligands to Ca2+, but helix and sheet secondary structures are slightly better providers of bidentate carboxylate ligation than turn or loop structures. The average coordination number for Ca2+ was 6.0, though for EF-hand sites it is 7. The average coordination number of a lanthanide ion in an intrinsic protein Ca2+ site was 7.2, but for the adventitious sites was only 4.4. A survey of the Cambridge Structural Database showed there are small-molecule lanthanide complexes with low coordination numbers but it is likely that water molecules, which do not appear in the electron density maps, are present for some lanthanide sites in proteins. A detailed comparison of the well-defined Ca2+ and lanthanide ion binding sites suggests that a reduction of hydrogen bonding associated with the ligating residues of the binding sites containing lanthanide ions may be a response to the additional positive charge of the lanthanide ion. Major structural differences between Ca2+ binding sites with weak and strong binding affinities were not obvious, a consequence of long-range electrostatic interactions and metal ion-induced protein conformational changes modulating affinities.     

Ion-binding to phospholipids. Interaction of calcium and lanthanide ions with phosphatidylcholine (lecithin).

Surface chemical and nuclear magnetic resonance (NMR) techniques have been used to study the interaction of Ca2+ and lanthanides with lecithins. With both methods positive reactions were detected at metal concentrations greater than 0.1 mM. 1H and 31P high-resolution NMR spectra obtained with single bilayer vesicles of lecithin were invariant up to Ca2+ concentrations of 0.1 M indicating that there is only a loose association between Ca2+ and the phospholipid. The weak interaction between Ca2+ and lecithin is confirmed by both surface chemical and NMR techniques showing that the packing of egg lecithin molecules present in bilayers does not change up to Ca2+ concentrations of about 0.1 M. The packing was also independent of pH between 1--10. Contradictory results have been reported in the literature concerning the question of Ca2+ binding to lecithins. The conflicting results are shown to have arisen from differences in the experimental conditions and differences in the sensitivity of the physical methods used by various authors to study Ca2+ -lecithin interactions. An estimate of the strength of binding and molecular details of the interaction were derived using paramagnetic lanthanides as isomorphous replacements for Ca2+. From the changes in chemical shifts induced in the presence of lanthanides an apparent binding constant KA approximately 30 l/mol was calculated at lanthanide concentrations greater than 10 mM. Using surface chemical methods it was shown that this KA is up to 10 times larger than that for Ca2+ binding. The complete assignment of the 1H NMR spectrum of lecithin, including the resonances from the relatively immobilized glycerol group, was determined to derive molecular details of the cation-lecithin interaction. From spin-lattice relaxation-time measurements and line broadening in the presence of GdCl3 it is concluded that the cations are bound to the phosphate group and that this is the only binding site. The absolute proton shifts induced by paramagnetic lanthanides depended on the nature of the ion, but the shift ratios standardised to the shift of the O3POCH2 (choline) signal were invariant throughout the lanthanide series indicating that the shifts are purely pseudocontact. In contrast the 31P shifts were found to contain significant contact contributions. These findings are consistent with a weak interaction and with the phosphate group being the binding site. The absolute shifts but not the shift ratios depended on the anion present indicating that the cation binding may be accompanied by binding of anions. Contrary to negatively charged phospholipids the interaction of lanthanides with lecithins was enhanced as the ionic strength was increased by adding NaCl. This was explained in terms of steric hindrance due to the extended conformation of the lecithin polar group.     

The effects of calcium and lanthanide ions on the activity of bovine heart nicotinamide adenine dinucleotide-specific isocitrate dehydrogenase

(i) The activity of purified NAD-specific isocitrate dehydrogenase from bovine heart was stimulated by free Ca²⁺ in the presence of ADP and subsaturating levels of magnesium isocitrate, but not in absence of ADP. However, Ca²⁺ was not absolutely required for ADP activation. This was particularly apparent when free Mg²⁺ was kept low (0.0024–0.020 mm) and the substrate magnesium dl-isocitrate ranged from 0.07–0.25 mm. When kinetic constants were determined at pH 7.4 under these conditions and in the absence of ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetate, Ca²⁺ had little or no effect on K_m (app) for ADP; the stimulation of rate by Ca²⁺ was mainly due to increased V (app). With subsaturating ADP, there was an interdependence in the interaction of the enzyme with substrate and Ca²⁺. Thus, with ADP constant (0.30 mm) the values of K_m (app) for magnesium dl-isocitrate declined from 0.35 mm at zero Ca²⁺ to 0.19 mm with saturating Ca²⁺ without affecting V; K_m (app) for free Ca²⁺ declined with increasing magnesium isocitrate to a limiting K_m of 0.3 μm. (ii) Ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetate, frequently used as a calcium buffer, inhibited enzyme activity with and without ADP. (iii) The enzyme was not inhibited by the calmodulin inhibitors trifluoperazine and chlorpromazine. Inhibition by lanthanide ions of the isocitrate dehydrogenase was competitive with magnesium isocitrate and not with respect to Ca²⁺. The values of K_is (1.8 to 3.1 μm) for La³⁺, Yb³⁺, Gd³⁺, Eu³⁺, Tb³⁺, and Er³⁺ were about two orders of magnitude smaller than K_m for magnesium dl-isocitrate.     

Evidence for the non-filamentous aggregation of actin induced by lanthanide ions.

We varied the molar ratio of added lanthanide ion to skeletal muscle actin (M3+/A) and observed their effects on the change in reduced viscosity (Nred) in the presence of polymerizing quantities of salt (0.1 M KC1). Once the concentration of the lanthanide ion exceeds the concentration of the nucleotide present (0.2 mM ATP), we noted that with M3+/A ratios up to 4: (a) there was a sharp peak in the observed Nred above the level achieved by control F-actin; (b) the magnitude of (a) was shown to be a function of the initial G-actin concentration. With an M3+/A ratio of greater than 4 we observed: (i) a sharp fall in the observed Nred; (ii) the formation of an insoluble aggregate of actin; (iii) the formation of (ii) was completely reversed by removal of the M3+; (iv) a complete inhibition of the ATP hydrolysis which always accompanies the G- to F-actin transition; (v) the number of mol of M3+ required to completely inhibit the rise in Nred (above the viscosity of G-actin) was a function of the ionic radii of the 11 lanthanide ions tested; and (vi) the effects described in (i) were not mimicked when the initial protein was in the F form. In the absence of added KCI, divalent cations (e.g. Mg2+) polymerize G-actin but this effect is not mimicked by the addition of the lanthanide ions. However, under these conditions the lanthanide ions cause the formation of an insoluble aggregate of actin. We conclude that with greater than 4 mol of lanthanide ions, G-actin aggregates in a form which contains little or no F-actin and that the lanthanide ion-induced aggregates are therefore different from the Mg2+-induced F-actin paracrystals.     

Cation selective promotion of tubulin polymerization by alkali metal chlorides.

A role for charge-based interactions in protein stability at the monomer or dimer level is well known. We show here that such interactions can also be important for the higher-order structures of microtubule assembly. Alkali metal chlorides increase the rate of polymerization of pure tubulin driven by either taxol or dimethyl sulfoxide. The effect is cation selective, exhibiting a sequence Na+ > K+ > Li+ > Cs+, with optimal concentrations for Na+ at approximately 160 mM. Hofmeister anion effects are additive with these rate stimulations. Sodium is less potent than guanidinium ion stimulation reported previously, but produces a larger fraction of normal microtubules. Alkali metal cations lower the critical concentration by a factor of approximately 2, produce cold reversible polymers whose formation is sensitive to podophyllotoxin inhibition, increase the fraction of polymers present as microtubules from approximately 0.9 to 0.99, and reverse or prevent urea-induced depolymerization of microtubules. In the presence of microtubule-associated proteins, the promotion of polymerization is no longer cation selective. In the polymerization of tubulin S, in which the acidic C termini of both monomers have been cleaved, the cation enhancement is markedly decreased, although selective persists. Because the selectivity sequence is similar to that of the coil/helix transition of polyglutamic acid, we suggest that a major part, although not all, of the cation selective enhancement of polymerization results from shielding of the glutamate-rich C termini of the tubulin monomers.     

The binding of calcium ions by erythrocytes and `ghost''-cell membranes

1. Washed human erythrocytes, suspended in iso-osmotic sucrose containing 2.5mm-calcium chloride, bind about 400mug-atoms of calcium/litre of packed cells. Sucrose may be replaced by other sugars. 2. Partial replacement of sucrose by iso-osmotic potassium chloride diminishes the uptake of calcium, 50% inhibition occurring at about 50mm-potassium chloride. 3. Other univalent cations behave like potassium, whereas bivalent cations are much more inhibitory. The tervalent cations, yttrium and lanthanum, however, are the most effective inhibitors of calcium uptake. 4. An approximate correlation exists between the calcium uptake and the sialic acid content of erythrocytes of various species and of human erythrocytes that have been partially depleted of sialic acid by treatment with neuraminidase. However, even after complete removal of sialic acid, human erythrocytes still bind about 140mug-atoms of calcium/litre of packed cells. 5. A Scatchard (1949) plot of calcium uptake at various Ca(2+) concentrations in the suspending media shows the presence of three different binding sites on the external surface of the human erythrocyte membrane. 6. Erythrocyte ;ghost' cells, the membranes of which appear to be permeable to Ca(2+) ions, can bind about 1000mug-atoms of calcium per ;ghost'-cell equivalent of 1 litre of packed erythrocytes. This indicates that there are also binding sites for calcium on the internal surface of the erythrocyte membrane.     

Transport of calcium ions by Ehrlich ascites-tumour cells.

Ehrlich ascites-tumour cells accumulate Ca2+ when incubated aerobically with succinate, phosphate and rotenone, as revealed by isotopic and atomic-absorption measurements. Ca2+ does not stimulate oxygen consumption by carefully prepared Ehrlich cells, but des so when the cells are placed in a hypo-osmotic medium. Neither glutamate nor malate support Ca2+ uptake in 'intact' Ehrlich cells, nor does the endogenous NAD-linked respiration. Ca2+ uptake is completely dependent on mitochondrial energy-coupling mechansims. It was an unexpected finding that maximal Ca2+ uptake supported by succinate requires rotenone, which blocks oxidation of enogenous NAD-linked substrates. Phosphate functions as co-anion for entry of Ca2+. Ca2+ uptake is also supported by extra-cellular ATP; no other nucleoside 5'-di- or tri-phosphate was active. The accumulation of Ca2+ apparently takes place in the mitochondria, since oligomycin and atractyloside inhibit ATP-supported Ca2+ uptake. Glycolysis does not support Ca2+ uptake. Neither free mitochondria released from disrupted cells nor permeability-damaged cells capable of absorbing Trypan Blue were responsible for any large fraction of the total observed energy-coupled Ca2+ uptake. The observations reported also indicate that electron flow through energy-conserving site 1 promotes Ca2+ release from Ehrlich cells and that extra-cellular ATP increase permeability of the cell membrane, allowing both ATP and Ca2+ to enter the cells more readily.     

The structural basis for the regulation of tissue transglutaminase by calcium ions.

The role of calcium ions in the regulation of tissue transglutaminase is investigated by experimental approaches and computer modeling. A three-dimensional model of the transglutaminase is computed by homology building on crystallized human factor XIII and is used to interpret structural and functional results. The molecule is a prolate ellipsoid (6.2 x 4.2 x 11 nm) and comprises four domains, assembled pairwise into N-terminal and C-terminal regions. The active site is hidden in a cleft between these regions and is inaccessible to macromolecular substrates in the calcium-free form. Protein dynamics simulation indicates that these regions move apart upon addition of calcium ions, revealing the active site for catalysis. The protein dimensions are consistent with results obtained with small-angle neutron and X-ray scattering. The gyration radius of the protein (3 nm) increases in the presence of calcium ions (3.9 nm), but it is virtually unaffected in the presence of GTP, suggesting that only calcium ions can promote major structural changes in the native protein. Proteolysis of an exposed loop connecting the N-terminal and C-terminal regions is linearly correlated with enzyme inactivation and prevents the calcium-induced conformational changes.     

Streptomyces viridochromogenes spore germination initiated by calcium ions.

Initiation of germination of heat-activated Streptomyces viridochromogenes spore occurs in media containing only calcium ions and organic buffer. The calcium-induced initiation of germination was accompanied by a decrease in absorbance of the spore suspension, an increased rate of endogenous metabolism, the loss of spore carbon, and the loss of heat resistance. Calcium amounts to 0.28% of the dry weight of freshly harvested spores. The amount of calcium remained the same after incubation of spores in water after heat activation. The spore content of calcium doubled after incubation in 0.5 mM CaCl2 for 5 min at 4 degrees C and during calcium-induced germination. Nearly all of the calcim appears to be bound to sites external to the spore membrane, since the chelating agents (ethylenedinitrilo) tetraacetic acid and arsenazo III removed virtually all of the calcium ions. The calcium ions must be present during the entire initiation of germination period. Germination ceases after an (ethylenedinitrilo) tetraacetic acid wash and begins again immediately after addition of calcium ions.