Binding of calcium ions to bacteriorhodopsin.

Adding Ca2+ or other cations to deionized bacteriorhodopsin causes a blue to purple color shift, a result of deprotonation of Asp85. It has been proposed by different groups that the protonation state of Asp85 responds to the binding of Ca2+ either 1) directly at a specific site in the protein or 2) indirectly through the rise of the surface pH. We tested the idea of specific binding of Ca2+ and found that the surface pH, as determined from the ionization state of eosin covalently linked to engineered cysteine residues, rises about equally at both extracellular and cytoplasmic surfaces when only one Ca2+ is added. This precludes binding to a specific site and suggests that rather than decreasing the pKa of Asp85 by direct interaction, Ca2+ increases the surface pH by binding to anionic lipid groups. As Ca2+ is added the surface pH rises, but deprotonation of Asp85 occurs only when the surface pH approaches its pKa. The nonlinear relationship between Ca2+ binding and deprotonation of Asp85 from this effect is different in the wild-type protein and in various mutants and explains the observed complex and varied spectral titration curves.     

Actin assembly by lithium ions.

The ability of Li+ to promote the assembly of actin has been compared with the more common cations used in actin assembly assays, K+, Mg2+, and Ca2+. The principal assay of actin assembly utilized was fluorescence photobleaching recovery (FPR), from which it is possible to determine the fraction of actin protomers incorporated into filaments and the average diffusion coefficients of the filaments. In addition, critical concentrations of actin over a range of concentrations of all of these cations have been determined using an assay that involves sonication and dilution of assembled actin filaments containing trace amounts of pyrene-labeled actin. The results demonstrate that Li+ is a more potent promoter of actin assembly than is K+. The more rapid assembly of actin in the presence of Li+ is attributable to an increased rate of filament elongation. Filaments assembled in equivalent concentrations of Li+ or K+ have the same diffusion coefficients, and thus presumably the same average lengths. The critical concentration of actin is about three times less in the presence of Li+ than in the presence of an equal concentration of K+. Cytochalasin D accelerates the rate of Li+-promoted actin assembly and reduces slightly the total fraction of actin assembly. However, cytochalasin D causes less shortening of filaments in the presence of Li+ than in the presence of K+ or Mg2+. By the criteria of assembly kinetics and critical concentration, Li+ is much less potent as a promoter of actin assembly than either Mg2+ or Ca2+. These results are discussed in terms of the role of electrostatic forces in the actin assembly mechanism and in terms of possible relationships to therapeutic and toxicity mechanisms for Li+.     

Interactions of metal ions with mu-monothiopyrophosphate.

mu-Monothiopyrophosphate (MTP) binds monovalent and divalent metal ions with dissociation constants (Kd) similar to those for pyrophosphate (PPi). The values of Kd for metal-MTP complexes are the following, as measured kinetically in the hydrolysis of MTP (microM): Mg2+, 32 +/- 4; Mn2+, 5.4 +/- 1.4; and Co2+, 27 +/- 15. The thermodynamically measured (EPR) values for Mg2+ and Co2+ are 28 +/- 13 microns and 11 +/- 4 microM, respectively; and the Kd for the complex MnPPi is 3.4 +/- 0.5 microM. The metal-MTP complexes undergo hydrolysis at rates modestly faster or slower than the rate at which MTP itself reacts. The complexes MgMTP2-, CoMTP2-, and MnMTP2- undergo hydrolytic cleavage with release of thiophosphate with observed first-order rate constants of 1.6 x 10(-2) min-1, 2.3 x 10(-2) min-1, and 0.6 x 10(-2) min-1, respectively, at 35 degrees C, compared with 1.1 x 10(-2) min-1 for MTP4- under the same conditions. Alkali metal cations also stimulate or retard the hydrolysis of MTP. At 25 degrees C and pH 12.2, the observed rate constant for tetramethylammonium MTP4- is 2.1 x 10(-3) min-1, and the estimated rate constants (min-1) for saturating alkali metals under the same conditions are as follows: Li+, 0.25 x 10(-3); Na+, 3.9 x 10(-3), K+, 6.7 x 10(-3); and Cs+, 6.7 x 10(-3). Divalent metal ions markedly retard the hydrolysis of MTP at pH 7 and 8 because complexation shifts the pH rate profile more than 2 pH units toward the acid side.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diffusional spread of iontophoretically injected ions.

Analysis of the Nernst-Planck equations governing movement of ions subjected to both a concentration gradient and an electric field shows that, under physiological conditions, electrophoresis plays no significant role in the spread of iontophoretically injected ions. Electrophoresis can be neglected in any region in which the specific resistance of the medium is substantially constant and where the electrical potential induced in the medium by the iontophoresis current is small compared to $\text{2RT}\text{F}$, or 50 mV.     

La ions in precipitated hydroxyapatites.

Hydroxyapatites were synthesized by precipitation from an aqueous solution with La3+ (0-0.75%) and with carbonate (0-6.1%) at controlled pH 7.0. Uptake of La3+ was 90-95% complete. Relatively low Ca/P (1.54-1.63) ratios were attributed to nonstoichiometry. Carbonate in samples was identified by IR spectroscopy as B-type carbonate. Lattice parameters of the hexagonal apatite structure were not affected by the La3+ content. Noncarbonated samples heated to 800 degrees C transform partially to beta-Ca3(PO4)2. Thermogravimetric analysis showed release of 0.4 mol adsorbed and 1 mol crystalline water up to 400 degrees C and decomposition of carbonate up to 900 degrees C in the samples. Luminescence data obtained for Gd-containing hydroxyapatites prove that Gd3+ ions are not incorporated in the precipitated hydroxyapatite. These findings suggest that, in the La-containing samples, La3+ is surface absorbed and not incorporated in hydroxyapatite.     

Displacement of sodium ions by surfactant ions from DNA. A 23Na-NMR investigation

23Na-NMR probes the ionic composition in the immediate vicinity of the DNA molecule, in the presence of a series of quaternary ammonium bromides, of varying hydrocarbon chain length. The 23Na-NMR line shows two Lorentzian components, in accordance with quadrupolar relaxation theory for S = 3/2 nuclei under slow modulation. Deconvolution of the observed lineshape provides, in a reliable manner, the relative fraction of sodium counterions neutralizing the phosphate sites on DNA. This quantity (p B chi 2) serves as an index of the relative affinities of various surfactant ions toward DNA, Na+ being the reference cation. The results are consistent with site binding of detergent ions to the nucleic acid, an interaction dominated by hydrophobic forces.     

Negative hydrophobic ions as transport-mediators for positive ions. Evidence for a carrier mechanism

The permeability of hydrophobic cations, such as tetraphenylarsonium across biological membranes and artificial lipid membranes is strongly increased in the presence of trace amounts of hydrophobic anions like tetraphenylborate (Liberman, Y.A. and Topaly, V.P. (1969) Biofizika 14, 452–461). Voltage-jump relaxation experiments performed on thin lipid membranes support the idea that the anions, A^?, act as carriers for the cations, B⁺, by the formation of neutral ion pairs, A^?B⁺. Their permeability is not affected by the electric dipole potential, which hinders the movement of free cations, B⁺.     

Cholinomimetic teratogens. II. Interaction with inorganic ions.

Treatment of chicken embryos after 96 h of incubation showed that the teratogenic activity of the cholinomimetic compounds carbachol, neostigmine, decamethonium, and tetramethylammonium is reduced in the presence of calcium acetate. Similarly, supplementation with potassium acetate reduced the teratogenicity of carbachol and tetramethylammonium.     

Inhibition of hydroxyproline synthesis by palladium ions.

Palladium ions, administered as PdSO4, markedly affect the incorporation of L-[3,4-3H2] proline into non-dialyzable fractions in 10-day chick embryo cartilage explants with a 55-65% reduction in the concentration range 0.06-0.6 mM. Under these conditions the synthesis of [3H]hydroxyproline was nearly completely inhibited. Experiments with prolyl hydroxylase (EC 1.14.11.2) indicated a strong irreversible inhibition of the enzyme with a competition between Fe2+ and Pd2+. The Ki for the inhibition was 0.02 mM. Pd2+-treated enzyme remained inactive after extensive dialysis. These studies suggest that Pd2+ may inhibit collagen synthesis by replacing Fe2+ in the active site of prolyl hydroxylase and forming strong complexes with the enzyme. These studies also point to a potential mechanism of Pd2+ toxicity.     

DNA hydrolysis by rare-earth metal ions.

Plasmid DNA and poly(dA) are cleaved by rare-earth(III) ions at pH 7-8 and 50 degrees C. The cleavage has been confirmed by prompt conversion of supercoiled pBR 322 plasmid DNA (Form I) to a relaxed Form II. Furthermore, degradation of poly(dA) to shorter oligonucleotides is clearly evidenced by HPLC. A possible application of the metal ions (and their complexes) to artificial nucleases is indicated.     

Hydrophobic carriers of vanadyl ions augment the insulinomimetic actions of vanadyl ions in rat adipocytes.

A novel family of vanadyl ion (VO2+, oxidation state +4) carriers is introduced. These carriers possess C2 symmetry, utilize two hydroxamate groups as ion binding sites, and optionally possess asymmetric carbons. Binding efficiencies and hydrophobicities are regulated by the use of a modular assembly. When applied to rat adipocytes, these carriers augment the potency of vanadyl ions to stimulate glucose metabolism. The complexes shift the dose-response curve to the left. Also, the maximal effect of vanadyl ions which is in the order of 20-30% of that of insulin is shifted toward maximal (100-115%) stimulation. Among several chelators studied, the order of synergistic potency was RL-252 greater than or equal to RL-262 greater than 1367. RL-239, RL-280, and RL-261 had smaller effects, whereas RL-282 had a negligible effect. The synergistic action of RL-252 (and other chelators as well) on VO2+ was already observed at a molar ratio of 1:0.01 of VO2+ to RL-252, respectively, and maximal augmentation occurred at a molar ratio of 1:0.1. The superiority of the hydrophobic chelators relative to the hydrophilic ones, together with the low molar ratio of chelator to VO2+ to achieve maximal effect, strongly suggests that these chelators act as vanadyl ionophores. This notion was confirmed by carrier-facilitated extraction of VO2+ from water into CHCl3 with the following order of decreasing efficacy: RL-262 greater than RL-252 greater than 1367 greater than RL-261. The chelators' potentiating effect may therefore be related to facilitated transport of VO2+ ions into the cells' interiors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Depolymerization of heparin by complexed ferrous ions.

Treatment of porcine heparin with the ferrous-EDTA complex and ascorbic acid for 24 h at 37 degrees C results in the degradation of most of the glycosaminoglycan to smaller fragments. About 65% of the products comprise oligosaccharides composed of less than 30 sugar units. The extent of depolymerization is decreased significantly if ascorbate or EDTA is not included in the reaction mixture. Gel filtration of the reaction products yielded fractions with narrow chain length ranges. The sulfate content of the fractions and their electrophoretic mobilities on cellulose acetate indicate that the components have equivalent charge densities. Depolymerization products with 20 or more sugar units retain significant anticoagulant potencies as measured by their effect in accelerating the neutralization of factor Xa by antithrombin.     

Transmembrane electrical currents of spin-labeled hydrophobic ions.

When spin-labeled phosphonium ions are rapidly mixed with phospholipid vesicles, time-dependent changes in the electron paramagnetic resonance spectrum of the spin label are observed. These changes are interpreted in terms of transmembrane transport of the hydrophobic ion, and simple analysis of the data at different membrane potentials is shown to give the binding constant of the ion to both membrane surfaces, the permeability, and current-voltage relationship for the vesicle membrane in the presence of the hydrophobic ion. These results establish the time resolution for methods using the phosphonium ion as a probe of time-dependent potentials across vesicle membranes, as well as provide fundamental information regarding the binding and transport of hydrophobic cations across bilayers. This latter point is significant in view of the fact that hydrophobic cations have not been well characterized in planar bilayers due to their weak binding and low conductance.     

Iron-sulphur clusters with labile metal ions.

A study has been carried out of the redox-linked metal ion uptake processes of the iron-sulphur cluster [3Fe-4S] in the bacterial ferredoxin, Fd III from Desulphovibrio africanus using a combination of electron paramagnetic resonance (EPR) and low-temperature magnetic circular dichroism (MCD) spectroscopy and direct, unmediated electrochemistry of the Fd in a film deposited at a pyrolytic graphite electrode. Reduction of the three-iron cluster is required before a divalent metal ion becomes bound as in the reaction sequence [formula: see text] The redox potentials of these processes and the metal binding constants have been determined. The affinities of the [3Fe-4S]0 cluster for divalent ions lie in the sequence Cd greater than Zn much greater than Fe. In addition, specific binding of a monovalent ion, Thallium(I), is detected for [3Fe-4S]1+ as well as for [3Fe-4S]0. The results provide a clear and quantitative demonstration of the capability of the open triangular tri-mu 2-sulphido face of a [3Fe-4S] cluster to bind a variety of metal ions if the protein environment permits. In each case the entering metal ion is coordinated by at least one additional ligand which may be from solvent (H2O or OH-) or from a protein side chain (e.g., carboxylate from aspartic acid). Hence the [3Fe-4S] core can be a redox-linked sensor of divalent metal ions, Fe(II) or Zn(II), that may trigger conformational change.     

Calcium ions and cell fusion. Effects of chemical fusogens on the permeability of erythrocytes to calcium and other ions.

1. Fusogenic and non-fusogenic chemicals were tesetd for their ability to allow 45Ca2+ and 3H2O to enter hen and human erythrocytes. 2. The ratio of 45Ca2+/3H2O in treated cells to that in untreated cells is referred to as the entry ratio. 3. Within 1 min at 37 degrees C both water-soluble and lipid-soluble fusogens increased the value of the entry ratio, which reached maximum values in 5--10 min. 4. Values of the entry ratio in the range of 4--12 were found under conditions that led to cell fusion. 5. Closely related but non-fusogenic chemicals did not significantly alter the entry ratio. 6. The entry ratios for 86Rb+, 22Na+ and 35SO42- were also significantly increased by both lipid-soluble and water-soluble fusogens, though the increases were not as large as those for 45Ca2+. 7. It is suggested that fusogenic compounds increase the permeability of biological membranes to ions, and that an increase in the concentration of intracellular Ca2+ initiates or facilitates events that lead to the chemically induced fusion of erythrocytes.