Metal chelates in the storage and transport of neurotransmitters: formation of mixed ligand chelates of Mg 2+ -ATP with biogenic amines

Abstract— Quantitative studies on the interactions of adenosine-triphosphate and several biogenic amines with magnesium ion have been carried out in an attempt to correlate the thermodynamic stabilities of the metal-binding of the amines with the in vivo affinities of the amines for granule-binding. Equilibrium data indicate that in each of the ternary chelate systems (viz. Mg²⁺-ATP-amine), the predominant reaction in the pH range 3.0–7.0 is the formation of a magnesium-ATP chelate with a stability constant, log K_ML=3.22 ± 0.02. Each of the biogenic amines coordinates with Mg²⁺-ATP system in the pH range 7.0–10.5 to form the mixed ligand chelate (or ternary chelate), Mg²⁺-ATP-amine(1:1:1). The stability constants for the binding of the amines with Mg²⁺-ATP are: (i) norepinephrine (NE) = 2.34 ± 0.32; (ii) epinephrine (E) = 2.95 ± 0.08; (iii) dopamine (DA) = 3.05 ± 0.06; (iv) octopamine (OA) = 1.93 ± 0.12; (v) 6-hydroxydopamine (6-HDA) = 2.42 ± 0.14; (vi) 3-methoxynorephedrine (MeN) =2.76 ± 0.09; (vii) amphetamine (AA) =2.09 ± 0.05; (viii) tyramine (TA) = 2.60 ± 0.04; (ix) phenylethylamine (PEA) = 0. A general correlation is indicated between the stability constants (binding strengths) of the amine chelates and the metal-binding functionalities of the amines on the one hand and their vesicular binding characteristics in in vivo systems on the other (Carlsson and Waldeck , 1966). The Mg²⁺-ATP-dependant amine storage mechanism of KIRSHNER (1962a;b) and Carlsson , Hillårp and Waldeck (1963) is discussed both in the light of the data on metal chelate stability and of a significant modification of metal coordination hypothesis.     

25Mg NMR study of Mg2+-ATP,ADP-creatine kinase complexes

²⁵Mg NMR spectroscopy was first applied to the ternary complexes consisting of Mg²⁺, ATP, ADP and creatine kinase. The ²⁵Mg NMR spectra of the Mg²⁺-ATP (or ADP) complex are remarkably broadened in the ternary Mg²⁺-ATP(or ADP)-creatine kinase complex in contrast with previous prediction. From temperature dependence of the spectra of the protein-bound ion, it is suggested that Mg²⁺ of the protein-bound Mg²⁺-ATP(or ADP) complex is not in the fast exchange regime. The ²⁵Mg NMR signal of the transition state analogue complex is narrower and less temperature-dependent than those of the ternary complex, suggesting that Mg²⁺ in the transition state analogue complex is in a more symmetrical environment or exchanges slower than that of the ternary complex.     

Effect of Adenosine and Intracellular GTP on KATP Channels of Mammalian Skeletal Muscle

We investigated the action of adenosine and GTP on K_ATP channels, using inside-out patch clamp recordings from dissociated single fibers of rat flexor digitorum brevis (FDB) skeletal muscle. In excised patches, K_ATP channels could be activated by a combination of an extracellular adenosine agonist and intracellular Mg²⁺-ATP and GTP or GTP-γ-S. The activation required hydrolyzable ATP and could be partially reversed with Mg²⁺, suggesting that it may involve a G-protein dependent phosphorylation of K_ATP channels. We found that K_ATP channels of the rat FDB could not be activated by Mg²⁺-ATP alone or by Mg²⁺-ATP in the presence of extracellular adenosine. Patches whose channel activity had been `rundown' by Ca²⁺ could not be recovered by adenosine, GTP or Mg²⁺-ATP. K_ATP channels activated by adenosine receptor agonists had a similar ATP sensitivity to those under control conditions; but adenosine appears to be able to switch these K_ATP channels from an inactive to an active mode. Received: 29 December 1995/Revised: 22 March 1996     

Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme

The chemical strategies used by ribozymes to enhance reaction rates are revealed in part from their metal ion and pH requirements. We find that kinase ribozyme K28(1-77)C, in contrast with previously characterized kinase ribozymes, requires Cu²⁺ for optimal catalysis of thiophosphoryl transfer from GTPγS. Phosphoryl transfer from GTP is greatly reduced in the absence of Cu²⁺, indicating a specific catalytic role independent of any potential interactions with the GTPγS thiophosphoryl group. In-line probing and ATPγS competition both argue against direct Cu²⁺ binding by RNA; rather, these data establish that Cu²⁺ enters the active site within a Cu²⁺•GTPγS or Cu²⁺•GTP chelation complex, and that Cu²⁺•nucleobase interactions further enforce Cu²⁺ selectivity and position the metal ion for Lewis acid catalysis. Replacing Mg²⁺ with [Co(NH₃)₆]³⁺ significantly reduced product yield, but not k_obs, indicating that the role of inner-sphere Mg²⁺ coordination is structural rather than catalytic. Replacing Mg²⁺ with alkaline earths of increasing ionic radii (Ca²⁺, Sr²⁺ and Ba²⁺) gave lower yields and approximately linear rates of product accumulation. Finally, we observe that reaction rates increased with pH in log-linear fashion with an apparent pKa = 8.0 ± 0.1, indicating deprotonation in the rate-limiting step.     

Free metal ion depletion by "Good's" buffers. I. N-(2-acetamido)iminodiacetic acid 1:1 complexes with calcium(ii). magnesium(II), zinc(II), manganese(II), cobalt(II), nickel(II), and copper(II).

Formation (affinity) constants for 1:1 complexes of N-(2-acetamido)iminodiacetic acid (ADAH₂) with Ca(II), Mg(II), Mn(II), Zn(II), Co(II), Ni(II), and Cu(II) have been determined. Probable structures of the various metal chelates existing in solution are discussed. Values for the deprotonation of the amide group in [Cu(ADA)] and subsequent hydroxo complex formation are also reported. The use of ADA as a buffer is considered in terms of metal buffers complexes which can be formed at physiological pH, i.e., at pH 7.0 there is essentially no free metal ion in 1:1 M²⁺ to ADA solutions.     

Copper(II), nickel(II) and zinc(II) complexes of N-acetyl-His-Pro-His-His-NH2: equilibria, solution structure and enzyme mimicking

The copper(II), nickel(II) and zinc(II) binding ability of the multi-histidine peptide N-acetyl-His-Pro-His-His-NH₂ has been studied by combined pH-potentiometry and visible, CD and EPR spectroscopies. The internal proline residue, preventing the metal ion induced successive amide deprotonations, resulted in the shift of this process toward higher pH values as compared to other peptides. The metal ions in the parent [ML]²⁺ complexes are exclusively bound by the three imidazole side chains. In [CuH₋₁L]⁺, formed between pH 6-8, the side chains of the two adjacent histidines and the peptide nitrogen between them are involved in metal ion binding. The next deprotonation results in the proton loss of the coordinated water molecule (CuH₋₁L(OH)). The latter two species exert polyfunctional catalytic activity, since they possess superoxide dismutase-, catecholase- (the oxidation of 3,5-di-tert-butylcatechol) and phosphatase-like (transesterification of the activated phosphoester 2-hydroxypropyl-4-nitrophenyl phosphate) properties. On further increase of the pH rearrangement of the coordination sphere takes place leading to the [CuH₋₃L]⁻ species, the deprotonated amide nitrogen displaces a coordinated imidazole nitrogen from the equatorial position of the metal ion. The shapes of the visible and CD spectra reflect a distorted arrangement of the donor atoms around the metal ion. In presence of zinc(II) the species [ZnL]²⁺ forms only above pH 6, which is shortly followed by precipitation. On the other hand, the [NiL]²⁺ complex is stable over a wide pH range, its deprotonation takes place only above pH 8. At pH 10 an octahedral NiH₋₂L species is present at first, which transforms slowly to a yellow square planar complex.     

Mechanistic origin of the kinetic cooperativity for the ATPase activity of sarcoplasmic reticulum

The (Ca²⁺-Mg²⁺)-ATPase from sarcoplasmic reticulum presents negative cooperativity for the hydrolysis of Mg²⁺-ATP at different concentration ranges of this substrate. A kinetic model is proposed according to which Mg²⁺-ATP may bind to three different enzymatic species present during the catalytic cycle, E (K ₁=1 µM), EP.Ca₂ (K ₉=500 µM) and *EP (K ₇=20 µM), accelerating the release of P_i. The fact that each of these species has a different affinity for Mg²⁺-ATP allows a significant enhancement of the rate of P_i release to the medium at the different ranges of Mg²⁺-ATP concentration where the enzyme shows a kinetic cooperativity. The kinetic analysis of this mechanism yields an equation which is a ratio of two cubic polynomials (3:3 rate equations) with respect to Mg²⁺-ATP and which may explain the negative cooperativity of the enzyme at different concentration ranges of Mg²⁺-ATP.Abbreviations: EGTA, ethylene glycol bis(-aminoethylether)-N,N,N,N-tetraacetic acid; I.U., international units; piruvate kinase (EC 2.7.1.40); lactate dehydrogenase (EC 1.1.1.27); ATP phosphohydrolase (EC 3.8.1.3).     

Metal ion inhibition of corn root plasma membrane atpase

In the presence of K⁺, the hydrolysis of ATP catalysed by the ATPase of corn plasma membrane showed negative cooperative kinetics. When the complexes of ATP and Mg²⁺, Mn²⁺, Ca²⁺ or Cd²⁺ were used as substrates, the catalysed hydrolysis changed to follow simple Michaelis-Menten kinetics. However, this change was not observed with Zn²⁺-ATP as the substrate. A substantial enhancement of the hydrolysis was observed only when the complexes of Mg²⁺ and Mn²⁺ were used. Kinetic parameter determination indicated that the enzyme exhibited a similar binding property but a different catalytic efficiency to Mg²⁺, Mn²⁺ and Ca²⁺-ATP. The enzyme formed a more stable but less reactive complex with Cd²⁺-ATP. The presence of aluminium ions competitively inhibited the membrane-catalysed hydrolysis of Mg²⁺-ATP, but showed no effect when free ATP was the substrate. This finding suggested that aluminium might bind in the vicinity of the Mg²⁺ of Mg²⁺-ATP in the active site of the enzyme. On the basis of these observed inhibitory effects, possible origins of metal ion toxicity to root plasma membrane ATPase activity are discussed.     

Cobalt(II), nickel(II), copper(II) and zinc(II) complexes with an open-chain pentadentate nitrogen ligand

The open-chain, potentially, pentadentate, ligan 1,11-bis(dimethylamino)-3,6,9-trimethyl-3,6,9,-triazaundecane (Me₇tetren) forms a series of metal complexes having the general formula [M(Me₇tetren)]Y₂ (Y = 1, M = Co, Ni; Y = ClO₄, M = Co, Ni, Cu, Zn). On the basis of their physical properties, it is suggested that all these compounds contains isostructural five-coordinate [M(Me₇tetren)]²⁺ cations, the ligand acting as pentadentate. These complexes react in solution with thiocyanate ion to give mono- and, with exception of copper(II), di-thiocyanato five- and six-co-ordinate derivatives. Mono-thiocyanato derivatives of cobalt(II), nickel(II) and zinc(II) have been isolated as tetraphenylborate salts. Cobalt(II) and nickel (II) di-thiocyanato derivatives have been also isolated. Results are discussed in terms of the steric requirements of the ligand and electronic properties of the metal ions.     

Laser flash photolysis studies of tris(2,2′-bipyridine)nickel(II) ion in aqueous solution

The photoreactions of tris(2,2′-bipyridine)nickel(II) complex, Ni(II)(bpy₃)²⁺ were studied and compared with that of the photoreactions of 2,2′-bipyridine ligand. Continuous photolysis of the complex shows that the photodecomposition corresponds to the absorption of light by the complex in the ligand centered excited state. Flash photolysis of the complex using a 248 nm excimer laser yields bipyridine cation radical and solvated electron as transients. Absorption spectra of the transient observed for the complex is found to be similar to that observed for the ligand on flash excitation using a 248 nm laser suggesting that the transients observed in the case of complex is due to the coordinated bipyridine in the complex. The formation of solvated electron is observed to be monophotonic and that of the bipyridine cation radical is found to be a biphotonic process. Significant change in Ni-N bond distance upon oxidation of Ni(bpy₃)²⁺ ion when compared to that observed in nickel(II)tetraazamacrocyclic complexes suggests that the formation of the trivalent complex by photolysis is not favored for the Ni(bpy₃)²⁺ ion and CTTS excited state in the complex is not observed in the present system.     

Evidence for two distinct Mg2+ binding sites in G(s alpha) and G(i alpha1) proteins

The function of guanine nucleotide binding (G) proteins is Mg²⁺ dependent with guanine nucleotide exchange requiring higher metal ion concentration than guanosine 5′-triphosphate hydrolysis. It is unclear whether two Mg²⁺ binding sites are present or if one Mg²⁺ binding site exhibits different affinities for the inactive GDP-bound or the active GTP-bound conformations. We used furaptra, a Mg²⁺-specific fluorophore, to investigate Mg²⁺ binding to α subunits in both conformations of the stimulatory (G_sα) and inhibitory (G_iα1) regulators of adenylyl cyclase. Regardless of the conformation or α protein studied, we found that two distinct Mg²⁺ sites were present with dissimilar affinities. With the exception of G_sα in the active conformation, cooperativity between the two Mg²⁺ sites was also observed. Whereas the high affinity Mg²⁺ site corresponds to that observed in published X-ray structures of G proteins, the low affinity Mg²⁺ site may involve coordination to the terminal phosphate of the nucleotide.     

Effects of chromium compounds on plasma membrane Mg2+-ATPase activity of BHK cells.

An ATPase activity stimulated by divalent ions (Mg²⁺, Ca²⁺, Mn²⁺, Zn²⁺) has been observed in intact hamster fibroblasts cultured in vitro (BHK line). Such activity has been determined by the incubation (30 min at 37°C) of washed cell suspensions (about 1 mg of proteins) in a medium containing 100 mM NaCl, 20 mM KCl, 15 mM Tris—HCl (pH 7.4), 10 mM NaHCO₃, 5 mM glucose and equimolar concentrations of ATP and divalent cation. Mg²⁺-ATPase activity is insensitive to ouabain and lacks specificity towards nucleoside triphosphate substrates. AMP and ADP are not hydrolyzed under these conditions. Apparent K_m of 0.76 mM and V_max of 1.46 μmol Pi · mg proteins^?1 · h^?1 have been calculated for Mg-ATP complex. This ATPase is an ectoenzyme, therefore its activity could be used as a suitable index of the action of chemicals like chromium compounds known for their cytotoxic effects on membrane functions.Salts of trivalent (CrCl₃) and hexavalent (K₂Cr₂O₇) chromium at concentrations ranging from 1 mM to 5 mM inhibit Mg²⁺-ATPase. The inhibition by K₂Cr₂O₇ is observed after pretreatment of the cells with this compound followed by its absence from the assay medium “per se” for Mg²⁺-ATPase, and it is referred to the alterations of membrane bound enzyme structures by the oxidizing hexavalent chromium. The inhibition by CrCl₃ is mainly evident when this compound is present in the incubation medium, and is referred to the interaction of trivalent chromium with Mg²⁺-ATP as it is partially reversed by increasing Mg²⁺-ATP concentration.     

Elucidation of the DNA-interacting properties and anticancer activity of a Ni(II)-coordinated mithramycin dimer complex

Mithramycin (Mith) forms a drug-metal complex with a 2:1 stoichiometry by chelation with a Ni(II) ion, which was determined using circular dichroism spectroscopy. Mith exhibits an increased affinity (~55 fold) for Ni(II) in the presence of DNA compared to the absence of DNA, suggesting that DNA acts as an effective template to facilitate chelation. Also, we characterized the DNA-acting properties of a Ni(II) derivative of Mith. Kinetic analysis using surface plasmon resonance and UV melting studies revealed that Ni^II(Mith)₂ binds to duplex DNA with a higher affinity compared to Mg^II(Mith)₂. The thermodynamic parameters revealed a higher free energy of formation for duplex DNA in the presence of Ni^II(Mith)₂ compared to duplex DNA in the presence of Mg^II(Mith)₂. The results of a DNA-break assay indicated that Ni^II(Mith)₂ is capable of promoting one-strand cleavage of plasmid DNA in the presence of hydrogen peroxide; the DNA cleavage rate of Ni^II(Mith)₂ was calculated to be 4.1 × 10^?4 s^?1. In cell-based experiments, Ni^II(Mith)₂ exhibited a more efficient reduction of c-myc and increased cytotoxicity compared to Mith alone because of its increased DNA-binding and cleavage activity. The evidence obtained in this study suggests that the biological effects of Ni^II(Mith)₂ require further investigation in the future.     

The crystallographic study of left-handed Z-DNA d(CGCGCG)2 and thermine complexes crystallized at various temperatures and at various concentration of cations

In crystals of complexes of thermine and d(CGCGCG)₂ molecules grown at 4, 10, and 20 °C, the numbers of thermine molecules connected to the DNA molecule were dependent on the temperature of the crystallization. Two molecules of thermine and one Mg²⁺ ion were connected to DNA molecule when thermine and d(CGCGCG)₂ were co-crystallized at 4 and at 20 °C. When an increased concentration of magnesium and thermine molecules were co-crystallized with d(CGCGCG)₂ molecules at 10 °C, three Mg²⁺ ions and only one thermine molecule were bound with a d(CGCGCG)₂ molecule. The number of polyamines and of Mg²⁺ ions connected to DNA was dependent on the atomic values of the polyamine and of the metal ion. The binding of more Mg²⁺ ions occurred when the atomic value of Mg²⁺ exceeded that of the corresponding mono- or polyamine, and when the Mg²⁺ ion concentration was elevated. Furthermore, this study is the first documentation of a naturally occurring polyamine bound to the minor groove of DNA in a crystal structure.     

Targeting topoisomerase II with the chiral DNA-intercalating ruthenium(II) polypyridyl complexes

Many antitumor drugs act as topoisomerase inhibitors, and the inhibitions are usually related to DNA binding. Here we designed and synthesized DNA-intercalating Ru(II) polypyridyl complexes Δ--[Ru(bpy)₂(uip)]²⁺ and Λ-[Ru(bpy)₂(uip)]²⁺ (bpy is 2,2′-bipyridyl, uip is 2-(5-uracil)-1H-imidazo[4,5-f][1,10]phenanthroline). The DNA binding, photocleavage, topoisomerase inhibition, and cytotoxicity of the complexes were studied. As we expected, the synthesized Ru(II) complexes can intercalate into DNA base pairs and cleave the pBR322 DNA with high activity upon irradiation. The mechanism studies reveal that singlet oxygen (¹O₂) and superoxide anion radical (O₂^•−) may play an important role in the photocleavage. The inhibition of topoisomerases I and II by the Ru(II) complexes has been studied. The results suggest that both complexes are efficient inhibitors towards topoisomerase II by interference with the DNA religation and direct topoisomerase II binding. Both complexes show antitumor activity towards HELA, hepG2, BEL-7402, and CNE-1 tumor cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Kinetics of acid-catalysed dissociation of lead(II) and copper(II) complexes of ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA)

The acid-catalysed dissociation rate constants for PbEGTA²⁻ and CuEGTA²⁻ complexes (where EGTA is ethylenebis(oxyethylenenitrilo) tetraacetic acid) were measured in acetic acid-acetate buffer medium (pH: 3.0–4.8) and perchloric acid solutions ([H⁺] = 0.05–0.15 M), respectively, at a constant ionic strength of 0.15 (NaClO₄). The rate laws shown by the lead(II) and copper(II) complexes are of the form, Rate = {k_d + k_H[H⁺]}[complex] and Rate = {k_d + k_H²[H⁺]²}[complex], respectively. Enthalpy and entropy of activation for acid-independent and acid-catalysed pathways for both the complexes were obtained by the temperature-dependence studies of resolved rate constants in the 16–45°C range. The rate of dissociation of PbEGTA²⁻ is not enhanced by increasing the concentration of acetate ion in the buffer, and the amount of total electrolyte in the reaction mixture has no pronounced effect on the dissociation rates of their the lead(II) or copper(II) complex. Attempts to study the kinetics of stepwise ligand unwrapping in the binuclear Cu₂EGTA complex were unsuccessful due to the extremely rapid dissociation of this complex to yield mononuclear CuEGTA²⁻.     

Equilibrium and structural studies on Co(II), Ni(II), Cu(II) and Zn(II) complexes with N-(2-benzimidazolyl)methyliminodiacetic acid: crystal structures of Ni(II) and Cu(II) complexes

Combined pH-metric, UV-Vis, ¹H NMR and EPR spectral investigations on the complex formation of M(II) ions (M=Co, Ni, Cu and Zn) with N-(2-benzimidazolyl)methyliminodiacetic acid (H₂bzimida, hereafter H₂L) in aqueous solution at a fixed ionic strength, I=10⁻¹ mol dm⁻³, at 25 ± 1 °C indicate the formation of M(L), M(H₋₁L)⁻ and M₂(H₋₁L)⁺ complexes. Proton-ligand and metal-ligand constants and the complex formation equilibria have been elucidated. Solid complexes, [M(L)(H₂O)₂] · nH₂O (n=1 for M = Co and Zn, n=2 for M = Ni) and {Cu (μ-L) · 4H₂O}_n, have been isolated and characterized by elemental analysis, spectral, conductance and magnetic measurements and thermal studies. Structures of [Ni(L)(H₂O)₂] · 2H₂O and {Cu(μ-L) · 4H₂O}_n have been determined by single crystal X-ray diffraction. The nickel(II) complex exists in a distorted octahedral environment in which the metal ion is coordinated by the two carboxylate O atoms, the amino-N atom of the iminodiacetate moiety and the pyridine type N-atom of the benzimidazole moiety. Two aqua O atoms function as fifth and sixth donor atoms. The copper(II) complex is made up of interpenetrating polymeric chains of antiferromagnetically coupled Cu(II) ions linked by carboxylato bridges in syn-anti (apical-equatorial) bonding mode and stabilized via interchain hydrogen bonds and π-π stacking interactions.     

“Off-On”switching electrochemiluminescence biosensor for mercury(II) detection based on molecular recognition technology

A novel “off-On” electrogenerated chemiluminescence (ECL) biosensor has been developed for the detection of mercury(II) based on molecular recognition technology. The ECL mercury(II) biosensor comprises two main parts: an ECL substrate and an ECL intensity switch. The ECL substrate was made by modifying the complex of Ruthenium(II) tris-(bipyridine)(Ru(bpy)₃²⁺)/Cyclodextrins-Au nanoparticles(CD-AuNps)/Nafion on the surface of glass carbon electrode (GCE), and the ECL intensity switch is the single hairpin DNA probe designed according to the “molecular recognition” strategy which was functionalized with ferrocene tag at one end and attached to Cyclodextrins (CD) on modified GCE through supramolecular noncovalent interaction. We demonstrated that, in the absence of Hg(II) ion, the probe keeps single hairpin structure and resulted in a quenching of ECL of Ru(bpy)₃²⁺. Whereas, in the presence of Hg(II) ion, the probe prefers to form the T-Hg(II)-T complex and lead to an obvious recovery of ECL of Ru(bpy)₃²⁺, which provided a sensing platform for the detection of Hg(II) ion. Using this sensing platform, a simple, rapid and selective “off-On” ECL biosensor for the detection of mercury(II) with a detection limit of 0.1 nM has been developed.     

Dinuclear copper(II) chloro complex of the ligand 2,3,5,6-tetra(2-pyridyl)pyrazine

Crystallographic and magnetic studies have been performed on the complex, [{CuCl}₂(μ-tppz)][PF₆]₂, where tppz is 2,3,5,6-tetra-2-pyridinylpyrazine. The crystal structure revealed an infinite, ionic chain wherein Cu(II) ions are respectively above and below the plane of the pyrazine moiety of the bridging tppz ligand with the pyridine moieties moving out of the pyrazine plane in order to coordinate to Cu(II). Each chloride ligand bonds equatorially to Cu(II) in a [{CuCl}₂(μ-tppz)]²⁺ ion and axially to a neighboring [{CuCl}₂(μ-tppz)]²⁺ ion so as to form a one-dimensional chain in the solid state. The temperature-dependent magnetic susceptibilitity could be satisfactorily fitted by using a modified Bleaney-Bowers expression (for H = −JS_a · S_bJ = −5.6 cm⁻¹ and g = 2.16) where the exchange interaction is suggested to involve the orbitals of the tppz ligand.