Thermodynamics of ATP hydrolysis from membrane electrode measurements of metal-ion ATP and ADP complexation

Free energies for the ATP-ADP hydrolysis reaction have been recalculated on the basis of new thermodynamic formation constants for metal-ion ATP and ADP complexes as determined by potentiometric measurements with ion-selective membrane electrodes. It is shown that free-energy maps are sharply altered at metal-ion levels greater than 10^?3m because of the effect of previously unrecognized 2:1 complexes of divalent metal ions with ATP and ADP. New formation constants for ADP complexes with sodium, potassium, calcium, and magnesium are also reported.     

13C nuclear magnetic resonance study of the complexation of calcium by taurine

¹³C Nuclear magnetic resonance chemical shifts, ¹J_C-C scalar coupling constants, spin-lattice relaxation times, and nuclear Overhauser effects were determined for taurine-[1, 2 ¹³C] and a taurine-[1 ¹³C] and taurine-[2 ¹³C] mixture in the presence and absence of calcium. Ionization constants for taurine amino and sulfonic acid groups and chemical shifts of N-methylene and S-methylene carbons of the taurine cation, zwitterion, and anion were obtained from simultaneous least squares analysis of ¹³C titration curves of both taurine carbons. Comparison of taurine titration shifts to values for related compounds reveals some unusual electronic properties of the taurine molecule. Stability constants of 1:1 calcium complexes with taurine zwitterions and anions, as well as their ¹³C chemical shifts, were obtained by least squares analysis of titration curves measured in the presence of calcium. The stability constants of calcium-taurine complexes were significantly lower than previous values and led to estimates that only approximately one percent of intracellular calcium of mammalian myocardial cells would exist in a taurine complex. The implications of these results with respect to the effect of taurine on calcium ion flux are discussed.     

Characterization of energy-dependent ca transport in maize mitochondria

Experimental conditions which optimize both substrate- and ATP-dependent Ca²⁺ transport in corn (Zea mays) mitochondria have been determined. It has been found that a substrate (pyruvate + succinate) dependent, Pi independent, binding of Ca²⁺ occurs. This reaction is very rapid and complete in less than 30 seconds. For massive accumulation of calcium, Pi is essential. Phosphate is accumulated along with the calcium and the ratio of Ca:Pi accumulated is about 1.6:1 indicating the precipitation of hydroxyapatite inside the mitochondria.

The activation energies and Michaelis constants for both the substrate- and ATP-driven reactions have been determined. It has also been shown that the substrate-driven system is more efficient in Ca²⁺ accumulation than the ATP-driven system. This is partially due to the fact that Mg²⁺ is essential for the ATP-driven system but not for the substrate-driven system and that Mg²⁺ acts as a strong competitor of Ca²⁺ transport. The effect of other inorganic ions on Ca²⁺ transport energized by both substrate and ATP were examined.

The results lend support to the hypothesis that high energy intermediates of oxidative phosphorylation participate directly in Ca²⁺ binding and transport in plant mitochondria.

     

The inhibition kinetics of yeast glutathione reductase by some metal ions

Glutathione reductase (GR, type IV, Baker's yeast, E.C 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). In this study some metal ions have been tested on GR; lithium, manganese, molybdate, aluminium, barium, zinc, calcium, cadmium and nickel. Cadmium, nickel and calcium showed a good to moderate inhibitory effect on yeast GR. GR is inhibited non-competitively by Zn^{2 +} (up to 2 mM) and activated above this concentration. Ca^{2 +} inhibition was non-competitive with respect to GSSG and uncompetitive with respect to NADPH. Nickel inhibition was competitive with respect to GSSG and uncompetitive with respect to NADPH. The inhibition constants for these metals on GR were determined. The chelating agent EDTA recovered 90% of the GR activity inhibited by these metals.     

Formation constants for interaction of citrate with calcium and magnesium ions

Formation constants for the interaction of citrate ion with calcium and magnesium ions in solution at 37°C and a constant ionic strength of 0.15 were determined by potentiometric titration. Values for the formation of CaL^? and CaHL⁰ complexes were 1.88 × 10³ and 67, respectively. Corresponding constants for MgL^? and MgHL⁰ were 2.19 × 10³ and 42, respectively. The existence of other complexes was not confirmed. Protonation constants were also determined under the same conditions.     

Terbium luminescence as a probe of the calcium binding site of trypsin and α-chymotrypsin

The large enhancement of the green luminescence of terbium ion which occurs on binding to porcine and bovine trypsins and to bovine α-chymotrypsin has been used to study the calcium binding sites of these enzymes. Excitation spectra, taken at low protein concentrations to minimize absorption effects, demonstrate that in each case, energy transfer occurs between the side chain of a tryptophan residue and bound Tb³⁺. Association constants for the binding of Tb³⁺ to the single binding site on each of the three proteins have been measured at 25 °C and pH 6.6. Ca²⁺ ions compete with Tb³⁺ for the single binding site, and association constants for Ca²⁺ were determined by Tb³⁺ displacement. The ratio of binding strengths of Ca²⁺ to α-chymotrypsin, bovine trypsin, porcine trypsin, and elastase is 1:12:24:23. Addition of Tb³⁺ to the homologous bacterial enzyme α-lytic protease caused no luminescence enhancement.     

Effects of calcium on the stability and activity of guinea pig liver transglutaminase

The binding of calcium to transglutaminase was studied by a kinetic method and by spectrophotometric titration. By the first method, we have shown that the binding of a single calcium per molecule, with a binding constant of 7500 +/- 1300 M-1 (at 55 degrees C), was responsible for the enhancement of the thermostability. The kinetic constants of the deactivation for the unliganded native form and the liganded native one are 1.47 +/- 0.04 min-1 and 0.32 +/- 0.05 min-1 respectively. The enhancement of thermostability is due to the stabilization of the native form, since the deactivation constants of the liganded and unliganded intermediate forms are equal. The total number of calcium binding sites, determined by titration is 4, and therefore, only 1 of them should be implicated in the thermostability. The 4 apparent association constants have been determined by a non-linear fitting of the data to the Adair equation. We have also shown a positive co-operative behaviour of the transglutaminase when the transferase is monitored versus calcium concentration.     

Calcium efflux and intracellular exchangeable calcium in mammalian nonmyelinated nerve fibers

Summary Calcium efflux was measured in desheathed rabbit vagus nerves loaded with⁴⁵Ca²⁺. The effects of extracellular calcium, sodium, phosphate, potassium and lanthanum ions on the calcium efflux were investigated and the distribution of intracellular calcium determined by kinetic analysis of⁴⁵Ca²⁺ efflux profiles. The⁴⁵Ca²⁺ desaturation curve can be adequately described by three exponential terms. The rate constant of the first component (0.2 min^–1) corresponds to an efflux from an extracellular compartment. The two slow components had rate constants of 0.03 and 0.08 min^–1 and represent the efflux from two intracellular pools. The amounts of exchangeable calcium in these two pools, after a loading period of 150 min, were 0.170 and 0.102 mmol/kg wet weight, respectively. The total calcium efflux in physiological conditions amounted to about 24 fmol cm^–2 sec^–1. The magnitude of the two intracellular compartments as well as the total calcium efflux were markedly affected by extracellular phosphate, sodium and lanthanum, whereas the corresponding rate constants remained almost unchanged. Phosphate reversed the effect of sodium withdrawal on the calcium efflux: in the absence of phosphate, sodium withdrawal increased the calcium efflux to 224%, but in the presence of phosphate, sodium withdrawal decreased calcium efflux to 44%. Phosphate also affected the increase in calcium efflux produced by inhibitors of mitochondrial calcium uptake, suggesting that two different mitochondrial pools contribute to the control and regulation of intracellular calcium and of the transmembrane calcium transport.Deceased 18 April 1988     

NMR Reinvestigation of the Caffeine–Chlorogenate Complex in Aqueous Solution and in Coffee Brews

Caffeine complexation by chlorogenic acid (3-caffeoylquinic acid, CAS Number [327-97-9]) in aqueous solution as well as caffeine–chlorogenate complex in freshly prepared coffee brews have been investigated by high-resolution ¹H-NMR. Caffeine and chlorogenic acid self-associations have also been studied and self-association constants have been determined resorting to both classical isodesmic model and a recently introduced method of data analysis able to provide also the critical aggregation concentration (cac). Furthermore, caffeine–chlorogenate association constant was measured. For the caffeine, the average value of the self-association constant determined by isodesmic model (K _i = 7.6 ± 0.5 M⁻¹) is in good agreement with the average value (K _a = 10 ± 1.8 M⁻¹) determined with the method which permits the determination of the cac (8.43 ± 0.05 mM). Chlorogenic acid shows a slight decreased tendency to aggregation with a lower average value of association constants (K _i = 2.8 ± 0.6 M⁻¹; K _a = 3.4 ± 0.6 M⁻¹) and a critical concentration equal to 24 ± 1 mM. The value of the association constant of the caffeine–chlorogenate complex (30 ± 4 M⁻¹) is compatible with previous studies and within the typical range of reported association constants for other caffeine–polyphenol complexes. Structural features of the complex have also been investigated, and the complex conformation has been rediscussed. Caffeine chemical shifts comparison (monomeric, complexed, coffee brews) clearly indicates a significant amount of caffeine is complexed in beverage real system, being chlorogenate ions the main complexing agents.     

A model for circular dichroism monitored dimerization and calcium binding in an EF-hand synthetic peptide.

EF-hand peptides have been shown to bind calcium and dimerize to form an intact protein domain [Shaw, G.S., Hodges, R.S. & Sykes, B.D. (1990). Science, 249, 280-283]. A synthetic 33-residue EF-hand peptide with the sequence of carp parvalbumin CD site demonstrated a seven-fold increase in the apparent calcium dissociation constant with a eight-fold decrease in peptide concentration when fit to a single-site calcium-binding model. This observation is consistent with EF-hand dimerization. This paper describes a method to determine the dimerization dissociation constant and the calcium dissociation constants for both the monomer and dimer forms of this EF-hand peptide using circular dichroism techniques. By monitoring the increase in negative molar ellipticity at 222 nm with increasing peptide concentration under calcium-saturating conditions the dimerization dissociation constant for the synthetic parvalbumin CD site was determined to be 55.68+/-10.76 microM. Using the dimerization constant, the calcium dissociation constants for both the monomer and dimer forms of this peptide were determined by monitoring the change in ellipticity of peptide solutions on addition of increasing amounts of calcium. A fit of this data to a mathematical model that takes into account dimerization results in calcium dissociation constants of 421.3+/-21.56 and 47.06+/-6.72 microM for the monomer and dimer forms, respectively.     

Interactions of calcium with the pyridine nucleotides

Association constants were determined for the 1:1 interactions of calcium with NAD⁺, NADH, NADP⁺, and NADPH in aqueous systems (pH 7, 25 °C) by use of a calcium-sensitive electrode. The order of binding of calcium to these pyridine nucleotides appears to be NAD⁺ < NADH < NADP⁺ < NADPH with association constants of 0.2 × 10², 0.3 × 10², 0.9 × 10², and 2 × 10², respectively. Calorimetric experiments revealed that all of these interactions are endothermic with enthalpy changes of 1, 2, 2, and 3 kcal/mol, respectively.     

The interactions between nucleic acids and polyamines. II. Protonation constants and 13C-NMR chemical shift assignments of spermidine, spermine, and homologs

Macroscopic protonation constants have been determined by pulentiometric titration for spermidine, spermine and for the four pulyamines. 3,5-Spd, 4,4-Spd, 4,3,4-Spm and 4,4,4-Spm. which are homologs of spermidine and spermine. A method for calculation of microscopic protonation constants of polyamines based on data for mono- and diamines gives results for spermidine that agree well with the experimental macroscopic protonation constants and the protonation sequence of Kimberly and Goldstein. ¹³C-NMR spectra of spermidine, spermine and six homologs have been obtained and used to assign specific resonances, correcting some ambiguity in the assignments for spermidine and some errors in the assignments for spermine.     

Spectrophotometric determination of reaction stoichiometry and equilibrium constants of metallochromic indicators: III. Antipyrylazo III complexing with Ca2+ and acetylcholine receptor protein

Stoichiometries, equilibrium constants and optical extinction coefficients of calcium-antipyrylazo III (An) complexing are determined with the analytical method described in article I of this series. Spcctrophotometric Ca titrations of An at the wavelengths 595 and 710 nm indicate overall dissociation equilibrium constants for the complexes CaAn, CaAn₂ and Ca₂An to be 4.5 × 10^?4 M, 1.1 × 10^?8 M² and 1.5× 10^?6 M², respectively, extrapolated to zero ionic strength. Ca titrations of solutions containing An plus acetylcholine receptor protein give clear evidence that An binds to the protein to a large extent in the presence of Ca²⁺; furthermore, addition of acetylcholine results in release of protein-bound Ca and An. This is the first reported indication that antipyrylazo III binds to biological material and questions the usefulness of this dye as a Ca indicator in biological systems.     

The pulse radiolysis pH-jump in aqueous solutions: applications to lanthanide(III)-methyl red systems

Use of the pulse radiolytic technique for studying acid-base and complex formation reactions in aqueous solutions is described. The method is based on the conversion of the hydrated electron e⁻_aq to the anion of a strong acid, thereby obtaining a fast pH-jump. Rate constants are calculated from relaxation times measured with either spectrophotometric or conductometric detection systems. The technique has been validated with previously studied chemical systems. Rate constants for the complex formation between La³⁺ and Gd³⁺ with Methyl Red indicator have also been determined.     

Speciation of ternary complexes of lead(II), cadmium(II) and mercury(II) with L-dopa and phenanthroline in propanediol-water mixtures

Abstract

The formation constants of ternary complexes of title systems have been determined pH-metrically in biologically relevant conditions at an ionic strength of 0.16 mol dm^-3 and 303 K. The overall stability constants have been evaluated using MINIQUAD75 computer program. The complexation equilibria have been derived on the basis of species distribution diagram. In the present study L-Dopa and 1, 10-phenanthroline are found to be compatible ligands, proving greater stability of ternary complexes as compared to binary ones. The trend in variation of stability constants with change in dielectric constant of medium is explained on the basis of electrostatic and non-electrostatic forces. Distributions of the species with pH at different compositions of propanediol-water mixtures are also presented. The factors responsible for the compatibility of both the ligands have also been discussed.     

Interaction of metal cations with Ca2+-transport sites of the plasma membrane Ca2+ pump of secretory cells of gastric glands

Investigation of Ca2+ transport by calcium pump of the cell plasma membrane of the gastric glands isolated from guinea pigs and its inhibition by metal cations has been performed. The mainly competitive type of Ca2+ translocation inhibition by the calcium pump by metals cations (0.025-1.00 mM) was determined. Potency of inhibition increases in such an order (I50, mM): Ba2+ (0.336) < Sr2+ (0.251) < Mn2+ (0.099) < Co2+ (0.029) < Cd2+ (0.016). It was shown by one-factor dispersion analysis that potency of inhibition depends on ionic radii and hydration enthalpy of metal cations and also on stability constants of their complexes with oxygen-containing bioligands (acetic, aspartic and glutamic acid) (hx2 = 83.73-85.95). Dependence of the inhibition constants (I50) on ionic radii is most adequately described by the parabolic equation, such a dependence on hydration enthalpy and stability constants with oxygen-containing bioligands--by exponential or multiplicative equations. The conclusion has been made that selective Ca2+ translocation by the calcium pump and its inhibition by metal cations is determined by the interaction between energy of their interaction with cation-binding sites of the transport system and energy of hydration. Energetics of such interactions depends on the steric factors. The physicochemical model of the Ca2+ selective translocation by calcium pump and its inhibition by metal cations has been proposed.     

Interaction of calcium ions with peptide hormones of the gastrin family

The interactions of Des-Trp¹-Nle¹²-minigastrin I (Nle¹¹-HG-13) and Nle¹⁵-little gastrin I (Nle¹⁵-HG-17) with calcium ions have been investigated in water and in trifluoroethanol solution using uv and CD absorption techniques. Both hormones strongly interact with Ca²⁺ in the organic medium. In the case of Nle¹¹-HG-13, the near-uv chiroptical properties (dominated by the transitions of the Trp residue in the C-terminal tetrapeptide sequence) indicate that three metal ions per mole of hormone are involved in the binding process. From the different response of near-uv and far-uv CD properties to the addition of calcium and from the change of the CD spectra in the aromatic absorption region, it is concluded that the biologically important C-terminal sequence is directly involved in the interaction with calcium. Elongation of the peptide chain from Nle¹¹-HG-13 to Nle¹⁵-HG-17 (Nle¹⁵-little gastrin I) does not provide any additional binding site for calcium ions. The change of the CD properties in the near- and far-uv indicates that three metal ions per mole of hormone are involved in the binding process. The dichroic absorption in the aromatic region indicates that only one of the two Trp residues of the little gastrin analog is sensitive to the presence of calcium. On the assumption that the variation of the CD properties is proportional to the extent of calcium binding, the binding constants K₁, K₂, and K₃ have been estimated roughly. Two similar sets of binding constants have been found, with K₁ ≥ 10⁶M^?1 and K₃ of the order of 10⁵M^?1, indicating similar binding sites in the two hormones with high affinity for calcium ions.     

Calcium chloride penetrates plant cuticles via aqueous pores

Penetration of calcium chloride across astomatous cuticular membranes (CMs) isolated from leaves of Pyrus communis L. has been studied. Penetration was a first-order process when calcium chloride concentrations ranged from 2 g l⁻¹ to 10 g l⁻¹. Rate constants were increased 10-fold by adding wetting agents but they did not depend on temperature. The accelerators tributyl phosphate and diethyl sebacate had no effect on rates of penetration. Increasing humidity over the salt residue on the CMs from 50 to 90% increased rate constants by about 2-fold. Extracting cuticular waxes from pear leaf CMs increased rate constants by factors of 2 to 3, depending on humidity. Leaf CMs from Malus domestica Borkh., Populus alba L., Stephanotis floribunda Brongn. and Schefflera actinophylla (Endl.) Harms were also permeable to CaCl₂. Highest rate constants were observed with poplar CMs while Schefflera CMs exhibited the lowest permeability. By comparing these results with the well established transport properties of the lipophilic pathway it is concluded that calcium chloride hexahydrate penetrated cuticular membranes via aqueous pores. Received: 14 December 1999 / Accepted: 31 March 2000     

Magnesium and lithium complexation by 1,4,7,10-tetraazacyclododecane

The stability constants of magnesium and lithium complexes of 1,4,7,10-tetraazacyclododecane (cyclen) have been determined in 0.5 M KNO ₃ at 25 °C by means of potentiometric titration, as K_MgL = 1.77 × 10 ² and K_{Li L} < 10 ⁻², respectively. Ab initio calculations on the protonated species of the cyclen ligand have been performed in order to obtain a better understanding of experimental protonation constants, and to compare them to previous calculations on the investigated metal complexes.