The role of metal ions in the activation of arginase.

The role of metal ions as activators of arginase hydrolyzing arginine were studied. The metal ion is assumed to form a complex with arginine and to promote the enzymatic reaction. The activating ability of the metal ion appears to be governed by the chelating ability and/or the coordination numbers which determine whether the metal ion combines with the enzyme or the substrate (or both substrate and enzyme) and factors which influence the configuration of the resulting complexes.     

Chelating peptide-immobilized metal ion affinity chromatography. A new concept in affinity chromatography for recombinant proteins

We report our experimental results supporting the hypothesis that a specific metal-chelating peptide (CP) on the NH2 terminus of a protein can be used to purify that protein using immobilized metal ion affinity chromatography (IMAC). The potential utility of this approach resides with recombinant proteins since the nucleotide sequence that codes for the protein can be extended to include codons for the chelating peptide and thereby generate the gene for a chimeric CP-protein that can be cloned, expressed, and affinity-purified with immobilized metal ions. The chelating peptide purification handle could then be removed chemically or enzymatically after purification has been achieved to generate a protein with the natural amino acid sequence. The feasibility of using a chelating peptide as a purification handle has been demonstrated using a leuteinizing hormone-releasing hormone (LHRH) analog, 2-10 LHRH, which contains the previously identified chelating peptide, His-Trp, on the NH2 terminus. 2-10 LHRH had a high affinity for a Ni(II) IMAC column due to the NH2-terminal dipeptide sequence His-Trp, forming a coordination complex with Ni(II), whereas the controls, 3-10 LHRH and 4-10 LHRH, lacking the CP sequence, did not bind. Furthermore, 2-10 LHRH could be purified from a mixture of histidine-containing peptides on a Ni(II) IMAC column in one step. His-Trp proinsulin was used as a model of a recombinant CP-protein. The S-sulfonates of His-Trp-proinsulin and proinsulin were isolated from Escherichia coli engineered to overproduce these proteins as trpLE' fusion proteins. His-Trp-proinsulin(SSO3-)6 had a higher affinity for immobilized Ni(II) than proinsulin (SSO3-)6. Both proteins were eluted by decreasing the pH or by introducing a displacing ligand into the buffer. Ni(II) eluted from the column with much higher concentrations of displacing ligand than the proteins.     

Famotidine, the new antiulcero-genic agent, a potent ligand for metal ions.

Potentiometric, polarographic, and spectroscopic results obtained for Cu2+ and Ni(2+)-famotidine systems clearly indicated that this anti-ulcerogenic drug is a very potent chelating agent able to coordinate cupric ion that was at pH below 2. This drug exhibits excellent histamine H2 receptor blocking effects and its effective coordination to metal ions may have significant biological implications. Famotidine is found to be a very effective ligand for Ni2+ ions also.     

Insulin-receptor interactions. Possible involvement of metals

Addition of Zn2+ or Cu2+ ions to plasma membrane preparations or to purified insulin receptors from rat liver resulted in an increase of specific insulin binding; no effect was observed with the addition of Fe3+, Ca2+ or Na+. Dialysis of membrane preparations, or of purified receptors, against chelating agents such as zincon (2-carboxy-2'-hydroxy-5'-sulfoformazyl-benzene) or 1,10-phenantroline resulted in a decrease in specific binding of insulin. With the readdition of Zn2+ or Cu2+ to the medium an increase in specific binding was observed, and values much higher than those of the original preparations were obtained; the addition of Ca2+, Fe3+ or Na+ to dialyzed preparations did not cause any effect on the specific binding. Dialysis of purified receptors against chelating agents resulted in a decrease in the content of Zn2+ and Cu2+. Zincon has been found to be a competitive inhibitor of insulin interfering with the specific binding to the receptor, and noncompetitive with the nonspecific binding. These results suggest the possible involvement of a metal ion present in the receptor in the formation of the insulin-receptor complex.     

Insights into the reaction of beta-lactam antibiotics with copper(II) ions in aqueous and micellar media: kinetic and spectrometric studies

Degradation of beta-lactam antibiotics by means of metallic cations seems to have a very complex chemistry, involving not only the catalytic effect of the metal ion but also complex formation. Many different compounds, such as methylpyrazines, oxazolones, penicilloic, penicillenic, and penicillonic acids, have been reported as degradation products of such antibiotics, although not many details about the progress of the reaction can be found in the literature. Two novel fluorimetric and spectrophotometric methods previously published by the authors, as well as kinetic studies, have been used to propose a possible reaction mechanism for the ampicillin degradation in the presence of copper(II) ions. Likewise, we have proposed the chemical structure required by the beta-lactam antibiotics to develop absorption or fluorescence properties. Kinetics in micellar and aqueous media shows that the copper-ampicillin reaction proceeds through different pathways depending on the reaction medium.     

Precipitation, chelation, and the availability of metals as nutrients in anaerobic digestion. I. Methodology

A methodology has been developed for the quantitative assessments of the individual effects of precipitation and chelation of metal ions in an anaerobic digester.     

The preparation of apo-Cu,Zn superoxide dismutase by ion-exchange chromatography on iminodiacetic acid-sepharose

The superoxide dismutases (EC 1.15.1.1) are a family of enzymes that catalyze the dismutation of superoxide radical anion to dioxygen and hydrogen peroxide. The active site contains a critical metal ion such as manganese, iron, or copper. The copper-containing protein also has one zinc ion bound per subunit. The standard method used to remove the metal ions from Cu,Zn superoxide dismutase has been to exhaustively dialyze the protein against chelating agents at low pH. We have developed a new method where the protein is bound to ion-exchange medium based on iminodiacetic acid immobilized on Sepharose. The bound protein is treated with a buffer containing edta at pH 3.5 to remove metal ions; the buffer is then exchanged for acetate buffer to remove edta, after which the protein is eluted by a salt gradient. An advantage of this method is that a single chromatography step is sufficient to produce apo protein. Results are shown for both human and bovine dimeric Cu,Zn superoxide dismutase and the monomeric Escherichia coli Cu,Zn superoxide dismutase. In every case, the metals were removed efficiently.     

The kinetics of flavine oxidation-reduction. II. Metal ion interactions.

The oxidation-reduction reactions of tetraacetylriboflavine in the presence of various metal ions in dimethylformamide have been investigated using the stopped-flow technique under anaerobic conditions. Dismutation kinetics in the presence of redox-inactive dissociated divalent metal ions such as Cd2+, Zn2+, and Fe2+ are typically triphasic. Metal ions act primarily upon an intermediate flavine dimer formed by fast association of flavoquinone and flavohydroquinone, resulting in a parallel formation and neutral and chelated radicals. A competition between metal ions and proton donors, e.g. the neutral flavohydroquinone (FredH3), is observed at the level of this intermediate complex. Small spectral changes occur secondarily as an ill-defined intermediate phase which could correspond to the reorganization of the solvation of radical chelate. The neutral radical is finally chelated at a much slower rate, the yield of total radical formation remaining almost unchanged during this kinetic phase. The oxidation of flavohydroquinone by ferric ions, either dissociated or strongly coordinated within a porphyrin, is complete and proceeds through biphasic kinetics. The first phase (Fred leads to F) is much faster than the second one (F leads to Fox). Dismutation resulting from the transient accumulation of neutral flavosemiquinone competes with the direct oxidation with ferric ions for the completion of the second oxidation step. The relative rate of dismutation is essentially limited by acidic-basic reactions in the absence of an excess of ferrous ion. The kinetic analysis of the direct oxidation reactions favors an outer-sphere mechanism for the electron transfer to the ferric ion, either free or strongly coordinated. The formation of a ferrous radical chelate can result from the dismutation reactions only when the amount of ferric ion initially present is not sufficient for complete oxidation.     

Aluminum effect on the activity of superoxide dismutase and of other antioxygenic enzymes in vitro

The effect of Al on superoxide dismutase (SOD) and on other antioxygenic enzymes: horseradish peroxidase, catalase, and glutathione peroxidase, has been investigated in vitro. In the case of SOD, the effect of metal chelators (EDTA and deferoxamine) and a possible synergistic effect with iron salts have also been tested using the pyrogallol assay. There is no significant inhibitory effect of Al on the activity of any of the above-mentioned enzymes. Noticeable increases in SOD activity were observed when metal chelators were added to the medium, but not when high concentrations of Al were present too, in the case of deferoxamine (DFO). The former fact seems to be a consequence of the chelation of transition metal ions that catalyze pyrogallol autoxidation by a mechanism not inhibitable by SOD, interfering in its action, which may account for part of the DFO antioxidant effect observed in vivo. The latter phenomenon could be owing to a saturation of the chelating capacity of DFO by an excess of Al present in the medium, which should bring the system back to the interfering conditions explained above. It can be concluded that Al, either in the presence or in the absence of iron salts, does not inhibit SOD activity in vitro. Moreover, no significant binding of Al to SOD was demonstrated, and the amounts of its metal constituents, Cu and Zn, were not affected by preincubation of the enzyme with Al. The effect of the different compounds tested on the rate of autoxidation of the indicating scavenger, pyrogallol, and a suitable hypothesis on their role in the oxidation process are also discussed.     

Interactions between different selenium compounds and zinc, cadmium and mercury

In this paper, we present a polarographic study of systems containing different inorganic and organic selenium compounds (sodium selenite, sodium selenate, seleno-methionine and seleno-urea) and metal ions (Zn2+, Cd2+ Hg2+) of the 12th group of elements in the periodic table. While zinc is a trace element known to be essential for plants and animals, cadmium and mercury are exogenous elements and are harmful pollutants that accumulate during aging; selenium is also recognized as an important micronutrient and is sometimes added to the diet. Experiments investigating the interactions were carried out using polarographic techniques in unbuffered systems. The three metal cations originated complexes with different strength and solubility in the presence of selenite anions; in the presence of selenate, polarography was not able to detect formation of complexes with these metal ions, at least under the experimental conditions used: a decrease of Hg2+ ion concentration was observed. Seleno-methionine did not react with Cd2+; in the presence of Zn2+, a soluble complex with a co-ordination number 1 was formed, while, again, the concentration of Hg2+ decreased in the presence of increasing concentrations of the selenium derivative. Seleno-urea did not react with Zn2+, but formed a complex with Cd2+ with limited solubility. Finally, this ligand could not be studied with Hg2+ because of the overlapping of the reduction potentials of both the ligand and the metal cation. Overall equilibrium constants for complex formation (Kf) and the solubility product (Ksp) for poorly soluble species are also reported.     

Preparation and characterization of chitosan-grafted-poly(2-amino-4,5-pentamethylene-thiophene-3-carboxylic acid N'-acryloyl-hydrazide) chelating resin for removal of Cu(II), Co(II) and Ni(II) metal ions from aqueous solutions

The graft copolymerization of ethylacrylate (EA) onto chitosan initiated by potassium persulphate and Mohr's salt combined redox initiator system in limited aqueous medium was carried out in heterogeneous media. Moreover, modification of the grafted chitosan was carried out by reaction of the ester group (-COOEt) with 2-amino-4,5-pentamethylene-thiophene-3-carboxylic acid hydrazide which eventually produce chitosan-grafted-poly(2-amino-4,5-pentamethylene-thiophene-3-carboxylic acid N'-acryloyl-hydrazide) (chitosan-g-ATAH) chelating resin. The application of the modified resin for metal ion uptake was studied using Cu(2+), Co(2+) and Ni(2+) ions. The modified chelating resins were characterized using FTIR spectroscopy, SEM and X-ray diffraction.     

Ecdysteroids act as inhibitors of calf skin collagenase and oxidative stress

Three new phytoecdysteroids have been isolated from the seeds of Chenopodium quinoa and structurally identified as 20,26-dihydroxy, 28-methyl ecdysone, 20,26-dihydroxy, 24(28)-dehydro ecdysone, and 20-hydroxyecdysone 22-glycolate using serial chromatographic and spectroscopic methods. Both previously reported compounds and newly identified phytoecdysteroids were evaluated for their inhibitory effect on calf skin collagenase, as this enzyme is involved in aging skin diseases. Their effectiveness on scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, as well as in chelating the iron metal ions was also investigated. All isolated compounds demonstrated a higher potency to inhibit this matrix metalloproteinase and to chelate the iron ion, both with respect to the number of carbonyl groups bearing their carbon skeleton, suggesting that their mechanism of action involves their ability to coordinate both ions (either the zinc ion of the catalytic domain of collagenase or the iron ion), acting as an electron donor. The DPPH-scavenging ability was hydroxyl dependent. Satisfactory results obtained from the enzyme in vitro experiment were further supported by the gel electrophoresis. These results indicate that ecdysteroids might be considered as potent chemical agents to prevent or delay both collagenase-related skin damages and oxidative stress.     

Neurotransmitters as ion shuttles in regulation of neural activity.

A molecular framework is described within which a single neural cell can modulate its excitability, or the quantity of transmitter released upon stimulation, in relationship to past stimulation. The key elements in this regulating system are complexation in the synaptic area by the transmitter molecule with extracellular ions, interaction of the complexed transmitter with the presynaptic receptor, possibly followed by reuptake of complexed transmitter.A number of mechanisms are suggested by which the transmitter/metal ion complex can regulate cellular function. Calculations are made to estimate the possible degree of change in the interior calcium concentration of a catecholaminergic cell by a calcium ion complex formed in the synapse. Experimental evidence is cited, which(a) documents the existence in the catecholaminergic cell of the necessary machinery for a calcium-ion-regulated cell-use registry device.(b) supports the hypothesis that catecholamines transport metal ions in neural systems, and(c) indicates that the ionic shuttle function of neurotransmitters plays a significant, but not exclusive, role in the transport of calcium. Calcium transported in this manner may be uniquely distinguishable from that derived from other sources of intracellular calcium in its temporal or spatial distribution. The existing evidence is discussed and rationalized with respect to the hypothesis that one of the chief presynaptic functions of many neurotransmitters is to feedback regulate cell function by performance as an ion shuttle.     

Effects of magnesium ions on the stabilization of RNA oligomers of defined structures

Optical melting was used to determine the stabilities of 11 small RNA oligomers of defined secondary structure as a function of magnesium ion concentration. The oligomers included helices composed of Watson-Crick base pairs, GA tandem base pairs, GU tandem base pairs, and loop E motifs (both eubacterial and eukaryotic). The effect of magnesium ion concentration on stability was interpreted in terms of two simple models. The first assumes an uptake of metal ion upon duplex formation. The second assumes nonspecific electrostatic attraction of metal ions to the RNA oligomer. For all oligomers, except the eubacterial loop E, the data could best be interpreted as nonspecific binding of metal ions to the RNAs. The effect of magnesium ions on the stability of the eubacterial loop E was distinct from that seen with the other oligomers in two ways. First, the extent of stabilization by magnesium ions (as measured by either change in melting temperature or free energy) was three times greater than that observed for the other helical oligomers. Second, the presence of magnesium ions produces a doubling of the enthalpy for the melting transition. These results indicate that magnesium ion stabilizes the eubacterial loop E sequence by chelating the RNA specifically. Further, these results on a rather small system shed light on the large enthalpy changes observed upon thermal unfolding of large RNAs like group I introns. It is suggested that parts of those large enthalpy changes observed in the folding of RNAs may be assigned to variations in the hydration states and types of coordinating atoms in some specifically bound magnesium ions and to an increase in the observed cooperativity of the folding transition due to the binding of those magnesium ions coupling the two stems together. Brownian dynamic simulations, carried out to visualize the metal ion binding sites, reveal rather delocalized ionic densities in all oligomers, except for the eubacterial loop E, in which precisely located ion densities were previously calculated.     

Combined chelation of bi-functional bis-hydroxypiridinone and mono-hydroxypiridinone: Synthesis, solution and in vivo evaluation

3-Hydroxy-4-pyridinones (3,4-HP) are well known iron-chelators with applications in medicinal chemistry, mainly associated with their high affinity towards trivalent hard metal ions (e.g. M³⁺, M = Fe, Al, Ga) and use as decorporating agents in situations of metal accumulation. The polydenticity and the extra-functionality of 3,4-HP derivatives have been explored, aimed at improving the chelating efficacy and the selectivity of the interaction with specific biological receptors. However, the ideal conjugation of both features in one molecular unity usually leads to high molecular weight compounds which can have crossing-membrane limitations.Herein, a different approach is used combining a arylpiperazine-containing bis-hydroxypyridone (H₂L¹) with a biomimetic mono-hydroxypyridinone, ornithine-derivative (HL²), to assess the potential coadjuvating effect that could result from the administration of both compounds for the decorporation of hard metal ions. This work reports the results of solution and in vivo studies on their chelating efficacy either as a simple binary or a ternary system (H₂L¹:HL²:M³⁺), using potentiometric and spectrophotometric methods. The solution complexation studies with Fe(III) indicate that the solubility of the complexes is considerably increased in the ternary system, an important feature for the metal complex excretion, upon the metal sequestration. The results of the in vivo studies with ⁶⁷Ga-injected mice show differences on the biodistribution profiles of the radiotracer, upon the administration of each chelating agent, that are mainly ascribed to the differences of their extra-functional groups and lipo/hydrophilic character. However, administration of both chelating agents leads to a more steady metal mobilization, which may be attributed to an improved access to different cellular compartments.     

Site-specific substituted cobalt(II) horse liver alcohol dehydrogenases. Preparation and characterization in solution, crystalline and immobilized state.

The specific substitution, using highly selective techniques, of catalytic and/or noncatalytic zinc ions by cobaltous ions in horse liver alcohol dehydrogenase (EC 1.1.1.1) has been studied with dissolved, crystalline and agarose-immobilised enzyme, in order to examine the effect of protein structure on the specificity of the metal exchange. The different binding sites can be clearly distinguished by the absorption spectra of their cobalt derivatives. In solution an anaerobic column chromatographic method made it possible to exchange half of the zinc in the enzyme by cobalt ions in a much shorter time than previous procedures. By raising the temperature in the exchange step, even the slowly exchanging zinc ions were substituted by cobalt, yielding products similar to cobalt alcohol dehydrogenases described earlier. Treatment of crystal suspensions of the enzyme with chelating agents (preferentially dipicolinic acid) gave an inactive protein with two zinc ions remaining bound. The enzyme could be reactivated by treatment of the crystalline protein with 5 mM zinc or cobaltous ions or by dialysis of dissolved inactive protein against 20 microM zinc or 1 mM cobaltous ions. Higher metal concentrations led to denaturation but the inactive protein could be crystallized from solution and then reactivated completely at higher metal concentrations. The preparation and absorption spectrum show that cobalt is bound specifically at the catalytic sites. Since metal substitution at these sites critically depends on the maintenance of the correct tertiary and quaternary structure, these must be preserved in the crystal lattice and partially altered in solution when the catalytic zinc ions are removed (or when excess of metal ions is applied), thus demonstrating the structure-stabilizing role of the catalytic metal ions. The enzyme immobilised on agarose, with unchanged content of active sites [Schneider-Bernl?hr et al. (1978) Eur. J. Biochem. 41, 475--484], was treated like the crystal suspensions. Although half of the zinc was removed, some activity remained. After reactivation with cobaltous ions, a loss of about 30% active sites was measured. Thus the apparently homogenous bound enzyme was rather heterogeneous in the properties of its catalytic metal binding sites. These results are taken as further proof for the dependence of the metal substitution on the proper tertiary and quaternary structure which is strained by multiple interactions in the covalently immobilised enzyme.     

Synthesis, metal ion binding, and biological evaluation of new anticancer 2-(2'-hydroxyphenyl)benzoxazole analogs of UK-1

UK-1 is a bis(benzoxazole) natural product displaying activity against a wide range of human cancer cell lines. A simplified analog of UK-1, 4-carbomethoxy-2-(2'-hydroxyphenyl)benzoxazole, was previously found to be almost as active as UK-1 against cancer cell lines, and similar to the natural product, formed complexes with a variety of metal ions such as Mg2+ and Zn2+. A series of 4-substituted-2-(2'-hydroxyphenyl)benzoxazole analogs of this 'minimal pharmacophore' of UK-1 were prepared. The anti-cancer activity of these analogs was examined in breast and lung cancer cell lines. Spectrophotometric titrations in methanol were carried out in order to assess the ability of UK-1 and these analogs to coordinate with Mg2+ and Cu2+ ions. Although none of the new analogs were more cytotoxic than 4-carbomethoxy-2-(2'-hydroxyphenyl)benzoxazole, some analogs were identified that display similar cytotoxicity to this simplified UK-1 analog with improved water solubility. UK-1 and all of these new analogs bind Cu2+ ions better than Mg2+ ions, and the nature of the 4-substituent is important for the Mg2+ ion binding ability of these 2-(2'-hydroxyphenyl)benzoxazoles. Previous studies of a limited number of UK-1 analogs demonstrated a correlation between Mg2+ ion binding ability and cytotoxicity; however, within this series of 4-substituted-2-(2'-hydroxyphenyl)benzoxazoles the variations in cytotoxicity do not correlate with either Mg2+ or Cu2+ ion binding ability. These results, together with recent ESI-MS studies of Cu2+-mediated DNA binding by UK-1 and analogs, indicate that UK-1 and analogs may exert their cytotoxic effects by interaction with Cu2+ or other transition metal ions, rather than Mg2+, and that metal ion-mediated DNA binding, rather than metal ion binding affinity, is important for the cytotoxic effect of these compounds. The potential role of Cu2+ ions in the cytotoxic action of UK-1 is further supported by the observation that UK-1 in the presence of Cu2+ displays enhanced cytotoxicity to MCF-7 and A549 cells when compared to UK-1 alone.     

Biotransformation of Heavy Metals from Soil in Synthetic Medium Enriched with Glucose and Shewanella sp. HN-41 at Various pH

The biotransformation of heavy metals from contaminated soil was examined using a facultative anaerobic bacterium Shewanella sp. HN-41. The experiments were carried out to assess the influence of glucose at various pH on the transformation of heavy metals from soil thorough solubilization. A preliminary study on the transformation of heavy metals from soil was first performed using a defined medium supplemented with glucose at 10, 20, and 30 mM to select the effective concentration. Among the three concentrations examined, glucose at 30 mM leached a highest level of metal ions. Therefore, 30 mM glucose was used as the representative carbon source for the subsequent experiments in a defined medium at various pH (5, 6, 7, 8, and 9). The organism HN-41 was not influenced by pH ranging from acidic to neutral and was able to metabolize all the metal elements from contaminated soil. The level of Fe, Cr, As, Mn, Pb, and Al solubilization ranged from 3 to 7664 mg kg^?1 at various initial pH. The rate of metal solubilization was found to be low at neutral pH compared with acidic and alkaline. These results are expected to assist in the development of heavy metal transformation processes for the decontamination of heavy metal-contaminated soil.