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.     

DNA interaction with biologically active divalent metal ions: binding constants calculation

In our previous work we have shown that under the action of Cu²⁺, Mn²⁺ and Ca²⁺ ions DNA is able to transit into a compact state in aqueous solution. In the present work we carried out calculations of binding constants for divalent metal ions interacting with DNA in terms of the macromolecule statistical sum. The formula for calculation of the binding constants and cooperativity parameters was proposed. It was shown that on the “coil state”–“compact (globule) state” transition a single DNA molecule may undergo the first-order phase transition while the transition of the assembly of average DNA chains is of sigmoidal character typical of the cooperative and continuous transition.     

Binding of divalent metal ions to calcium-free peroxidase: thermodynamic and kinetic studies

Thermodynamics of binding of divalent metal ions including Ca(2+) , Mg(2+) , Ba(2+) , and Cd(2+) to Ca-free horseradish peroxidase (HRP) enzyme was investigated using UV/VIS spectrophotometry and molecular-mechanic (MM) calculations. According to the obtained binding and thermodynamic parameters, trend of the relative binding affinities of these divalent metal cations was found to be: Ca(2+) >Cd(2+) >Mg(2+) >Ba(2+) . Binding analysis based on Scatchard and Hill models showed positive cooperativity effect between the two distal and proximal binding sites. Furthermore, kinetics of binding and reconstitution process was examined (using relaxation-time method) for binding of Ca(2+) (as the typical metal ion) to Ca-free HRP, which was found a second-order type having a two-step mechanism involving fast formation of Ca-free HRP/1?Ca(2+) as the kinetic intermediate in step 1. Finally, by means of MM calculations, the comparative stability energies were evaluated for binding of M(2+) metal cations to Ca-free HRP. Based on MM calculations, preferential binding of Ca(2+) ion was occurred on distal and proximal binding sites of Ca-free HRP associated with higher stability energies (E(total) ). Indeed, among the divalent metal ions, Ca(2+) with the highest binding affinity (maximum value of K(bin) and minimum value of ΔG$\rm{{_{bin}^{0}}}$), maximum value of exothermic binding enthalpy, and stability energies stabilizes the HRP structure along with an optimized catalytic activity.     

Interaction of gastrin with transferrin: effects of ferric ions

The sedimentation behavior of 125I-labeled gastrin has been studied as a function of Fe3+ ion concentration and pH. Both sedimentation velocity and sedimentation equilibrium experiments indicated that high-molecular-weight Fe3+-gastrin complexes were formed at pH 5.0 and pH 7.4. Self-association of gastrin alone was observed at pH values below 5.0. 125I-labeled gastrin bound to human serum apotransferrin at pH 7.4. Scatchard analysis of the gastrin-apotransferrin complex gave a Kd of approximately 6.4 microM at 37 degrees C, with two binding sites per molecule of apotransferrin. No significant binding of gastrin to diferric transferrin was observed under the same conditions. The binding of gastrin to apotransferrin was inhibited by NaCl. The results are consistent with the hypothesis that gastrin and transferrin act synergistically in the uptake of dietary iron by the gastrointestinal tract.     

Interaction of mithramycin with metal ions and DNA

The interaction of mithramycin with metal ions has been studied by absorbance and fluorescence spectroscopy. Magnesium shifts the drug absorbance spectrum to longer wavelengths and displays a weak binding constant (Kd = 1mM); no interaction with calcium was detected. The drug requires magnesium for binding to DNA and this is characterised by small additional hypochromic and bathochromic changes. Mithramycin does not bind to DNA in the presence of calcium. With 10mM magnesium the drug binds to DNA with an association constant of 9.2 x 10(4) M-1. The inability of calcium to substitute for magnesium has been confirmed by 'footprinting' experiments using both DNase I and hydroxyl radicals.     

Interaction of metallic cations with DNA VI. Specific binding of Mg++ and Mn++

The binding of Mg²⁺ and Mn²⁺ by DNA by a divalent cation specific electrode and by ultracentrifugation. Both techniques give similar results for the stoichiometry of the reaction. An oscillating densiemete allowed us to detect small changes of volume accompanying the binding. The reaction was also followed by circular dichroism measurements. Interpretation of the results is only possible if one assumes an electrostate site-binding of Mg²⁺ to phosphate group, and a chelation Mn²⁺ between the phosphate group and the N₇ of the guanine. Physical modifications accompanying these two types of binding are discused and compared to the role of these cations in some biological systems involving DNA.     

Structure-function studies on gastrointestinal hormones: I. Synthesis of secretin analogs and their biological and immunological properties

Syntheses by conventional procedures of the three analogs corresponding to the porcine secretin sequence crossed at position 6 by the N-terminal hexapeptide sequences of VIP, GIP, and glucagon are described, viz., Ala⁴,Val⁵-, Tyr¹,Ala²,Glu³-, and Gln³-secretin (VIP-SN, GIP-SN, and GLU-SN). The analog Phe¹,Phe²,Trp³,Lys⁴-secretin (SOMA-SN), designed on the basis of the surprising homology of the sequence portions 10–13 of somatostatin and 5–8 of secretin, was also prepared. Finally, the synthesis of N^α-3-(4-hydroxyphenyl)propionyl-β-alanyl-secretin (DATA-SN), a tracer suitable for secretin radioimmunoassay and as an N-terminus modified secretin analog, is reported. The analogs are compared, in terms of their biological and immunological properties in different assay systems, with pure synthetic secretin.     

Interaction of nitrogenase with nucleotide analogs of ATP and ADP and the effect of metal ions on ADP inhibition

The interaction of a large number of ATP and ADP analogs with nitrogenase from Azotobacter vinelandii, Klebsiella pneumoniae, and Clostridium pasteurianum has been examined. Only 1,N6-etheno-ATP and 2'-deoxy-ATP served as substrates for acetylene reduction. Other triphosphates including GTP, ITP, 8-Br-ATP, alpha,beta-methylene ATP, beta,gamma-methylene ATP, 6-chloropurine riboside triphosphate, and AMP-PNP were inert, showing less than 50% inhibition at levels up to two- to fivefold greater than ATP. Xanthosine triphosphate behaved simply as a chelator of magnesium, activating the enzyme at low levels but strongly inhibiting at high levels. When nucleotide diphosphates were tested as inhibitors with enzyme from A. vinelandii, GDP, dGDP, and 6-chloropurine riboside diphosphate were ineffective, XDP was three- to fivefold less effective, and dADP and 1,N6-etheno-ADP were about equally as effective as ADP. With enzyme from C. pasteurianum, dADP was twofold less effective than ADP, XDP was fivefold less effective, and IDP and 1,N6-etheno-ADP appeared to be ineffective. Results with enzyme from K. pneumoniae were very similar to those obtained with A. vinelandii. Different metal ions were tested in the presence of both ATP and ADP to determine whether preferential binding to one nucleotide or the other might alter the ADP/ATP ratio needed for 50% inhibition of activity. Magnesium and manganese gave the same ratio, while with Fe and Co, slightly less ADP was required for equivalent inhibition. Nickel appeared to reduce the sensitivity of A. vinelandii nitrogenase to ADP inhibition while increasing that of C. pasteurianum, but both effects were less than twofold. Calcium, strontium, and aluminum ions were inert with enzymes from these organisms. Cd and Zn were also ineffective with K. pneumoniae. Two isomers of ATP beta S were prepared by enzymatic synthesis from ADP beta S. The A form was a more potent inhibitor of A. vinelandii nitrogenase.     

Comportment of S. cerevisiae in relation to ions Ca++ and Mg++ on beet molasses wort

Summary Calcium and magnesium are present in molasses at variable and sub-optimal concentrations for yeast metabolism. A molasse wort containing 60 p.p.m. of magnesium and 125 p.p.m. of calcium allows an optimal fermentation. If calcium becomes quickly toxic by accumulation in the yeasts, this effect can be corrected by additions of magnesium, without significant effect on the biomass.     

Binding of transition metal ions to albumin: Sites,affinities and rates

Background

Serum albumin is the most abundant protein in the blood and cerebrospinal fluid and plays a fundamental role in the distribution of essential transition metal ions in the human body. Human serum albumin (HSA) is an important physiological transporter of the essential metal ions Cu^2 +, and Zn^2 + in the bloodstream. Its binding of metals like Ni^2 +, Co^2 +, or Cd^2 + can occur in vivo, but is only of toxicological relevance. Moreover, HSA is one of the main targets and hence most studied binding protein for metallodrugs based on complexes with Au, Pt and V.

Scope of Review

We discuss i) the four metal-binding sites so far described on HSA, their localization and metal preference, ii) the binding of the metal ions mentioned above, i.e. their stability constants and association/dissociation rates, their coordination chemistry and their selectivity versus the four binding sites iii) the methodology applied to study issues of items i and ii and iv) oligopeptide models of the N-terminal binding site.

Major Conclusions

Albumin has four partially selective metal binding sites with well-defined metal preferences. It is an important regulator of the blood transport of physiological Cu(II) and Zn(II) and toxic Ni(II) and Cd(II). It is also an important target for metal-based drugs containing Pt(II), V(IV)O, and Au(I).

General Significance

The thorough understanding of metal binding properties of serum albumin, including the competition of various metal ions for specific binding sites is important for biomedical issues, such as new disease markers and design of metal-based drugs. This article is part of a Special Issue entitled Serum Albumin.     

The effect of divalent metal ions on the activity of Mg++ depleted ribulose-1,5-bisphosphate oxygenase

Ribulose-1,5-bisphosphate carboxylase-oxygenase is deactivated by removal of Mg⁺⁺. The enzyme activities can be restored to a different extent by the addition of various divalent ions in the presence of CO₂. Incubation with Mg⁺⁺ and CO₂ restores both enzyme activities, whereas, the treatment of the enzyme with the transition metal ions (Mn⁺⁺, Co⁺⁺, and Ni⁺⁺) and CO₂ fully reactivates the oxygenase: however, the carboxylase activity remains low. In experiments where CO₂-free conditions were conscientiously maintained, no reactivation of RuBP oxygenase was observed, although Mn⁺⁺ ions were present. Other divalent cations such as Ca⁺⁺ and Zn⁺⁺, restore neither the carboxylase nor the oxygenase reaction. Furthermore, the addition of Mn⁺⁺ to the Mg⁺⁺ and CO₂ preactivated enzyme significantly inhibited carboxylase reactions, but increased the oxygenase reaction.Abbreviation RuBP ribulose-1,5-bisphosphate. The enyme unit for RuBP carboxylase is defined as mol CO₂ fixed·min^-1 and for the RuBP oxygenase as mol O₂ consumed · min^-1     

The binding of Mg++ ions to polyadenylate, polyuridylate, and their complexes

The binding of Mg ⁺⁺ to polyadenylate (poly A), Polyuridylate(poly U), and their complexes, poly (A + U) and poly (A + 2U), was studied by means of a technique in which the dye eriochrome black T is used to measure the concentration of free Mg^?. The apparent binding constant K_X = [MgN]/[Mg⁺⁺][N], N = site for Mg⁺⁺ binding (the phosphate group of the nucleotide), was found to decrease rapidly as the extent of binding increased and, at low extents of binding, as the concentration of Na^? increased in poly A, poly (A + U), and poly (A + 2U), and somewhat less so in poly U. K_x is generally in the range 10⁴ > K_X > 10². The cause of these dependences is apparently, primarily, the displacement of Na⁺ by Mg⁺⁺ in poly U and poly (A + U) on the basis of the similarity of extents of displacement measured in this work and those measured potentiometrically. was calculated and was found to approach zero as the concentration of Na⁺ increased. In poly U, poly (A + U), and poly (A + 2U) at low ΔH′ v.H. > 0, about + 2 kcal/“mole.” In poly A, also at low salt, ΔH′ v.H. ≈ ?4 kcal/“mol” for the initial binding of Mg⁺⁺, and increases to +2 kcal/“mol” at saturation. This enthalpic variation probably accounts for the anticooperativity in the binding of Mg⁺⁺ not ascribable to the displacement of Na⁺⁺.     

Binding of Mg2+ to single-stranded polynucleotides: hydration and optical studies

The binding of Mg(2+) to single-stranded ribo- and deoxy-polynucleotides, poly(rA), poly(rU), poly(dA) and poly(dT), has been investigated in dilute aqueous solutions at pH 7.5 and 20 degrees C. A combination of ultrasound velocimetry, density, UV and CD spectroscopy have been employed to study hydration and spectral effects of Mg(2+) binding to the polynucleotides. Volume and compressibility effects of Mg(2+) binding to random-coiled poly(rU) and poly(dT) correspond to two coordination bonds probably between the adjacent phosphate groups. The same parameters for poly(rA)+Mg(2+) correspond to an inner-sphere complex with three-four direct contacts. However, almost no hydration effects are arising in binding to its deoxy analog, poly(dA), indicating mostly a delocalized binding mode. In agreement with hydration studies, optical investigations revealed almost no influence of Mg(2+) on poly(dA) properties, while it stabilizes and aggregates poly(rA) single-helix. The evidence presented here indicates that Mg(2+) are able to bind specifically to single-stranded polynucleotides, and recognize their composition and backbone conformation.     

Interaction of NAD(H)-dependent dehydrogenases with active dyes and their complexes with transitional metal ions

The interaction of NAD(H)-dependent dehydrogenases--yeast alcohol dehydrogenase and rabbit muscle lactate dehydrogenase--with reactive dyes produced in the USSR was studied. The essential role of metal ions in specific binding of alcohol dehydrogenase and dyes was demonstrated by differential spectroscopy, circular dichroism spectroscopy and chromatography. Lactate dehydrogenase in contrast with alcohol dehydrogenase does not require metal ions for the binding of the above-said dyes. A comparative study of eluting abilities of selected desorption agents (imidazole, adenine, 8-oxyquinoline-5-sulfonic acid, NAD, AMP, EDTA) by alcohol dehydrogenase chromatography on adsorbents with light-resistant yellow 2KT-Cu(II) and orange 5K revealed the differences in competition of the dyes for NAD-binding sites of alcohol dehydrogenase. The participation of light-resistant yellow 2KT-Cu(II) in the formation of mixed complexes with imidazole, adenine, 8-oxyquinoline-5-sulfonic acid, NAD and EDTA suggests that the specific binding of alcohol dehydrogenase to light-resistant yellow 2KT-Cu(II) is due to coordination between the Cu(II) ion and the amino acid residue in alcohol dehydrogenase.     

Interaction of DNA with heavy metal ions and polybases: cooperative phenomena

The polarographically determined binding constants of Cu⁺⁺ and Cd⁺⁺ to DNA were found to decrease with the degree of binding. The binding causes a reduction in the electrostatic potential ψ on the DNA molecule. The binding constant conforms to the relation K = K₀ exp {-zeψ/kT}. The binding constant at zero potential K₀ depends on the nature of the competing counterfoils to the phosphate group of the DNA; it is apparently smaller when Na⁺ rather than the ethylpyridonium residue of polyvinylpyridine serves as the competing counterfoil. The specific interact ion between the ethylpyridonium residues of the polybase and the DNA is very weak, even though the polyelectrolytic interaction induced by the decrease of the electrostatic free energy is spectacular. The intereaction is reversible and equilibrium is maintained between the different interaction products. A reshuffling of the interacting partners can be effected by spontaneous or induced (by centrifugal forces) precipitation of the least soluble interaction products participating in the equilibrium.     

Binding of ions to chitosan—selectivity studies

Selectivity coefficients for binding of negative and positive ions to chitosans of different chemical composition have been determined by equilibrium dialysis. Chitosans with different fraction of acetylated units (F_A of 0.01 and 0.49) generally behaved similarly in their selectivity towards both negative and positive ions. No selectivity was found in the binding of chloride and nitrate ions, while chitosan showed a strong selectivity towards molybdate polyoxyanions, with selectivity coefficients around 100. Chitosan showed a strong selectivity towards copper (Cu²⁺) compared to the metal ions zinc (Zn²⁺), cadmium (Cd²⁺) and nickel (Ni²⁺), with selectivity coefficients from 10 to 1000, while little or no selectivity could be detected with the other metal ions. Ionic strength and pH did not influence the selectivity coefficients of the chitosans towards the metal ions.