Evidence of metal binding activities of pentadecapeptide from Panax ginseng

A tetradecapeptide from ginseng (Panax ginseng) root showing anti-lipolytic activity in an isolated rat fat cell assay was chemically synthesized for analysis of metal binding activities in vitro. Binding activities against several metal ions were analysed by measuring mobility shifts during capillary zone electrophoresis experiments. The ginseng polypeptide (GPP) showed the greatest increase in effective molecular electrophoretic mobility in the presence of Mg²⁺. Mobility was also affected in the presence of La³⁺, Mn²⁺, Ca²⁺ and Zn²⁺ ions. Analysis with the dye Stains-all revealed GPP to possess a cation binding site similar to those in Ca²⁺-binding proteins. GPP thus appears to be a metal binding peptide. The results of this analysis suggested that GPP may perform its anti-lipolytic activities through an ability to modulate the level of free cellular Mg²⁺ and Mn²⁺ ions.     

Thematic minireview series: Metals in Biology 2012

Metals are present in about one-half of the protein structures available and also have critical roles in nucleic acid biochemistry. This prologue introduces the fourth of the Thematic Minireview Series on Metals in Biology, which deals with several topics involving iron, manganese, copper, and other metals. The six minireviews discuss metal transport and intracellular homeostasis, including chaperones and siderophores, maturation of the diiron active sites in hydrogenases, the balance between manganese and iron, and copper homeostasis relevant to pathogens.     

油茶微量元素铜铁锌吸收和积累特征

为弄清油茶(Camellia oleifera)对微量元素铜、铁、锌的吸收利用特征,对5年生‘长林4号’各器官的铜、铁、锌元素含量及其年变化进行了研究。结果表明,油茶植株中铁元素的含量最高,其次为锌和铜元素;单株油茶对锌、铜元素的年积累量分别为62.97 mg和22.60 mg,约为3∶1。从果实发育期至成熟期,锌元素的单株吸收积累量为40.18 mg,约占年吸收积累量的63.81%,从抽梢期至果实成熟期,铜元素的单株吸收积累量为20.04 mg,占年吸收积累量的88.67%,从休眠期至抽梢期,油茶地上部分生长所需的铜、锌元素分别有30.25%和57.90%来源于根系贮存的营养;油茶对铁元素的吸收积累则集中在抽梢期至果实发育期,单株吸收量达0.34 g。这些为指导油茶科学施肥提供了理论依据。     

Micronutrient uptake and distribution in mycorrhizal or phosphorus-fertilized soybeans

Summary Soybean plants were grown in a soil very low in available P. Seedlings were inoculated with two vesicular-arbuscular mycorrhizal (VAM) fungi or were left non-inoculated and fertilized with P. Assimilation and allocation of micronutrients (Fe, Mn, Zn, and Cu) were determined during host development, and the uptake of trace elements in VAM plants was compared to P-fertilized, non-VAM plants of similar weight, growth stage, and P status. Copper and zinc concentrations were always higher in VAM plants, while iron and manganese concentrations were lower than in the equivalent P-fertilized soybeans. Differences in the micronutrient content of fully-mature soybean pods reflected differences in the leaves and roots. Thus, for trace elements, seed quality can be altered by VAM colonization in a fashion not duplicated by P fertilizer. Contribution from the Western Regional Research Center, USDA-ARS (CRIS No. 5325-20580-003).     

Protein metalation in biology

Inorganic metals supplement the chemical repertoire of organic molecules, especially proteins. This requires the correct metals to associate with proteins at metalation. Protein mismetalation typically occurs when excesses of unbound metals compete for a binding site ex vivo. However, in biology, excesses of metal-binding sites typically compete for limiting amounts of exchangeable metals. Here, we summarise mechanisms of metal homeostasis that sustain optimal metal availabilities in biology. We describe recent progress to understand metalation by comparing the strength of metal binding to a protein versus the strength of binding to competing sites inside cells.     

A method for controlling the activities of free metal,hydrogen, and phosphate ions in hydroponic solutions using ion exchange and chelating resins

Summary An ion exchange and chelating resin system was developed to buffer the activities of selected free cations and phosphate in hydroponic solutions at concentrations similar to those that occur naturally in soil solutions. Free-ion activities of Cd, Cu, Ni, Zn, Mn, H, Ca, Mg, and K were maintained by ion exchange and chelating resins in a controlled ionic strength system. Iron was controlled by EDDHA and chelating resin, and P by a cation-exchange resin containing adsorbed polynuclear hydroxyaluminum. This mixed-resin hydroponic system was used to establish a range of ionic Cd activities similar to that found in soil solutions of soils amended with sewage sludge. Activities of other nutrients were maintained at realistic soil-solution levels. A metal complexing agent (EDTA) was used to increase total metal concentration in the hydroponic solutions without significantly altering the ionic activities of the metals maintained in solution. This allowed differentiation of the effects of free ions and complexed metals on metal uptake. Concentrations of metal complexes in solution were controlled by the ion activities of the metals maintained and the concentrations and selectivities of the complexing agent. The mixed-resin system supplied sufficient nutrients for the growth of tomato plants (Lycopersicon esculentum, cv. Wisconsin-55) in hydroponic culture. Research supported by the College of Agricultural and Life Sciences, University of Wisonsin-Madison and by United States Environmental Protection Agency through Grant CR807270010.     

Binding abilities of dehydropeptides towards Cu(II) and Ni(II) ions. Impact of Z–E isomerization on metal ion binding

The study on the binding ability of dehydro-tri- and tetrapeptides has shown that the α,β-double bond has a critical effect on the peptide coordination to metal ions. It may affect the binding of the vicinal amide nitrogens by the electronic effect and stabilize the complex due to steric effects. The (Z) isomer is the most effective in stabilizing of the complexes formed. The presence of large side chain in the dehydroamino acid residue may also be critical for the coordination mode in the metallopeptide systems.     

Transition metal binding to cod otolith proteins

Otolith microchemistry can be very useful in identifying fish populations and reconstructing fish movements. Recent attempts have been made to evaluate otoliths as proxies of ambient levels of transition metals, but findings have been inconsistent. Some of the difficulty with obtaining a definitive answer stems from an incomplete understanding of the biological control of transition metal speciation in otoliths. Metals may be incorporated as part of the calcium carbonate phase, trapped in interstitial spaces within the crystal, or associated with the protein matrix. Metal binding to the protein phase may be inferred from its structural and biochemical properties but has not been observed previously. Inherent difficulties with the extraction of metal-binding proteins in their native state from the calcium carbonate phase make them extraordinarily difficult to measure. We have developed a method that facilitates the extraction of otolith proteins without total disruption of transition metal binding. Chelating agents such as EDTA, used in the decalcification of otoliths, can demetallate the proteins if allowed to reach equilibrium; however, if the reaction is halted prior to equilibration, intact metal-protein complexes can be obtained. Using such an approach, we have confirmed the presence of copper and zinc in the soluble portion of the protein matrix of cod otoliths, and we have established that between 70% and 100% of copper and 40% to 60% of zinc found in whole otoliths are associated with the soluble part of the protein matrix. Manganese was not observed to be associated with the protein, indicating that it is either weakly bound or that no binding is present. Our results, combined with an understanding of the biological control of these metals, suggest that otoliths are not likely to be reliable indicators of copper and zinc exposure, but they may provide useful insight into fish growth and physiological development.     

Metal binding to alpha-synuclein peptides and its contribution to toxicity

Recent studies have suggested that alpha-synuclein (AS) is a metal binding protein. Metals also induce protein aggregation. In order to clarify controversy over the location of the metal binding sites six peptide fragments spanning the full length of the protein were analysed to identify metal binding domains. Our results indicated that both the C-terminus of the protein and a region around histidine 50 play a role in copper binding. We suggest that the true binding domain is a nonlinear site composed of both areas acting together to bind copper. The toxicity of these peptides to SH-SY5Y cells was also studied. There was a copper-independent component associated with the NAC domain of the protein and a copper-dependent component associated with the C-terminus of the protein and potentiated by involvement of the N-terminus. We hypothesise that copper binding can cause conversion of AS to a neurotoxic form via inter-protein interactions.     

Boron,copper, iron,manganese and zinc contents in leaves of flowering sunflower plants (Helianthus annuus,L.), grown with different boron supplies

Summary Boron, copper, iron, manganese and zinc concentrations were measured in sunflower leaves from plants grown hydroponically in a closed continuous flow system and with boron concentrations ranging from deficient to toxic. Leaves were analyzed at the stage of flower development. There was a highly significant inverse correlation between B concentration in solution and Mn concentration in leaves. Cu, Fe and Zn concentrations in the leaves were not changed by the different B levels, although the B concentration in leaves increased with an increasing concentration in solution, showing a close correlation each other.     

Iron and copper in plasma membranes of maize (Zea mays L.) roots investigated by proton induced X-ray emission

Summary Plasma membranes of maize (Zea mays L., cv. Sil Anjou 18) roots were isolated by aqueous two-phase partitioning. Multi elemental analysis by proton induced X-ray emission (PIXE) was used for the investigation of elemental composition of plasma membranes. Fe, Cu, and Zn as well as P, S, and Ca were identified. We did not find significant amounts of V, Mn, Se, Mo, or W.Abbreviations EDTA ethylenediaminetetraacetic acid - HCF III hexacyanoferrate III (ferricyanide, K₃[FeCN₆]) - Hepes 2-[4-(2-hydroxyethyl)-1-piperazine]-ethanesulfonic acid - PIXE proton induced X-ray emission (proton microprobe) - STA siliciotungstic acid - Tris tris (hydroxymethyl)aminomethane     

Hints for Metal-Preference Protein Sequence Determinants: Different Metal Binding Features of the Five Tetrahymena thermophila Metallothioneins

The metal binding preference of metallothioneins (MTs) groups them in two extreme subsets, the Zn/Cd- and the Cu-thioneins. Ciliates harbor the largest MT gene/protein family reported so far, including 5 paralogs that exhibit relatively low sequence similarity, excepting MTT2 and MTT4. In Tetrahymena thermophila, three MTs (MTT1, MTT3 and MTT5) were considered Cd-thioneins and two (MTT2 and MTT4) Cu-thioneins, according to gene expression inducibility and phylogenetic analysis. In this study, the metal-binding abilities of the five MTT proteins were characterized, to obtain information about the folding and stability of their cognate- and non-cognate metal complexes, and to characterize the T. thermophila MT system at protein level. Hence, the five MTTs were recombinantly synthesized as Zn²⁺-, Cd²⁺- or Cu⁺-complexes, which were analyzed by electrospray mass spectrometry (ESI-MS), circular dichroism (CD), and UV-vis spectrophotometry. Among the Cd-thioneins, MTT1 and MTT5 were optimal for Cd²⁺ coordination, yielding unique Cd₁₇- and Cd₈- complexes, respectively. When binding Zn²⁺, they rendered a mixture of Zn-species. Only MTT5 was capable to coordinate Cu⁺, although yielding heteronuclear Zn-, Cu-species or highly unstable Cu-homometallic species. MTT3 exhibited poor binding abilities both for Cd²⁺ and for Cu⁺, and although not optimally, it yielded the best result when coordinating Zn²⁺. The two Cu-thioneins, MTT2 and MTT4 isoforms formed homometallic Cu-complexes (major Cu₂₀-MTT) upon synthesis in Cu-supplemented hosts. Contrarily, they were unable to fold into stable Cd-complexes, while Zn-MTT species were only recovered for MTT4 (major Zn₁₀-MTT4). Thus, the metal binding preferences of the five T. thermophila MTs correlate well with their previous classification as Cd- and Cu-thioneins, and globally, they can be classified from Zn/Cd- to Cu-thioneins according to the gradation: MTT1>MTT5>MTT3>MTT4>MTT2. The main mechanisms underlying the evolution and specialization of the MTT metal binding preferences may have been internal tandem duplications, presence of doublet and triplet Cys patterns in Zn/Cd-thioneins, and optimization of site specific amino acid determinants (Lys for Zn/Cd- and Asn for Cu-coordination).     

Pinus radiata D. Don in Chile. Fertilization trials with copper

Summary YoungPinus radiata D. Don were fertilized with copper oxychloride and copper sulfate on a random block basis with four replicates. Roots, needles, stems and shoot apices were analyzed separately for Cu, Fe, Mn and Zn contents at different times from fertilization. The results showed a rapid plant copper absorption, no effects being found on the Fe, Mn and Zn content. The dynamic internal distribution of copper among different plant parts is shown. The treatments applied did not cause any phenotypical alteration nor phytotoxicity in the plants, and no significant effect of the fertilizer chemicals on each other.     

Displacement of frequently occurring heacy metals in autumn leaves of beech (Fagus sylvatica)

Summary By utilizing energy-dispersive X-ray microanalysis the heavy metals manganese, iron, zinc and lead were determined in different tissues of beech (Fagus sylvatica L.) autumn leaves. It was observed that all four metals were accumulated in sieve element-companion cell complexes of the leaf veins in the period from the green (2 October) to the brown (23 October) leaf coloration. Concomitantly, the concentration of these metals decreased in certain tissues of the stems subtending the leaves. The major reservoirs of heavy metals in the stems were periderm, cortex, pith and xylem rays. Since sieve element-companion cell complexes of the stems showed increasing metal concentrations during the transition from the green to the brown leaf stage, and since this temporary increase also occurred in the petiole phloem, it is inferred that symplastic transport can be used in addition to apoplastic transport for the displacement of heavy metals. It is assumed that the accumulation of heavy metals in the veins of autumn leaves indicates the deposition of surplus ions which are excluded when leaves are cast off.Supported by Deutsche Forschungsgemeinschaft and by Bundesministerium für Forschung und Technologie, Federal Republic of Germany, Grant PBE 373607.     

Effect of rhizosphere pH on the availability and uptake of Fe,Mn and Zn

In pot experiments the relationships between rhizosphere pH, the extractable levels of Fe, Mn and Zn in the soil and their uptake into the roots and shoots of dwarf French beans have been studied. Variations in rhizosphere pH were induced by applying three different sources of nitrogencholine phosphate, ammonium phosphate and calcium nitrate-to an initially homogenised soil (preadjusted to either pH 7 or 8). The rhizosphere pH was found to be significantly lower following the application of either ammonium or choline phosphates and to be increased by calcium nitrate treatment.The Fe and Zn contents of both shoot and root were inversely proportional to rhizosphere pH. The Mn contents also increased with decreasing pH but a sharp increase was apparent below pH 5.5. The shoot Fe, Zn and Mn content were significantly correlated with the extractable levels determined in the rhizosphere and non-rhizosphere soil.This paper forms part of the Ph. D. thesis submitted by A.N.S. to the University of Wales in 1977.     

The interaction of biological and noxious transition metals with the zinc probes FluoZin-3 and Newport Green

Zinc-sensitive fluorescent probes have become increasingly important in the investigation of the cellular roles of zinc. There is, however, little information on how the other transition metals in cells may influence the measurement of zinc. We have characterized in vitro the interaction of the nominal zinc indicators FluoZin-3 and Newport Green with all the cationic transition metals found within cells, Cr, Mn, Fe, Co, and Cu, as well as Ni and Cd, by measuring their dissociation constants. In addition, we have shown how FluoZin-3 can be used to quantify the concentration of copper in a cell-free assay and report that the fluorescence of Newport Green is boosted by both Cu(I) and Fe(II). Furthermore, we have introduced diagnostics for detecting the interference of metals other than zinc with its measurement within cells.     

The parallel expression of metal tolerance in pollen and sporophytes of Silene dioica (L.) Clairv., S. alba (mill.) krause and Mimulus guttatus DC

Summary The purpose of this paper was to determine if heavy metal tolerance was expressed in pollen and if its expression was correlated with the tolerance of the pollen source. Clones of Silene dioica, tolerant to zinc, closely related but nontolerant S. alba and clones of Mimulus guttatus tolerant and sensitive to copper were grown in the greenhouse in either standard potting soil or nutrient culture. Pollen was collected shortly after dehiscence, hydrated, and tested over a broad range of metal concentrations. The tolerance of the pollen source was determined by comparing root growth in solutions with and without heavy metals. In both Silene species and M. guttatus, the tolerance of the parental clone was expressed in its pollen. Pollen from tolerant individuals was able to germinate and grow at concentrations of metals which markedly inhibited pollen from nontolerants.     

Metal binding and antioxidant properties of chimeric tri- and tetra-domained metallothioneins

An unusual tri-domained (alpha-beta-beta) natural oyster metallothionein (MT) is known, and non-oxidative MT dimers occur in vivo in mollusk species and in mammals. To assess the respective role of the MT domains, two chimeric MTs were constructed: a tetra-domained oyster MT corresponding to the alpha-beta-alpha-beta structure, in order to mimic the natural non-oxidative dimeric form, and a tri-domained alpha-beta-alpha oyster MT. Metal binding and putative antioxidant properties of these two chimeric MTs were investigated using expression of the related genes in the bacteria Escherichia coli. In a wild-type strain these MTs could efficiently bind Cd. In a superoxide dismutase (sodA sodB) null mutant, the tri-domained MT was found to exacerbate Cd toxicity whereas the tetra-domained MT efficiently protected bacteria from Cd. The paradoxical toxicity displayed by the tri-domained MT upon Cd contamination was linked to the generation of superoxide radicals generated by a mechanism which most probably involves a copper-redox cycling reaction, since a Cd-contaminated sodA sodB strain expressing this MT produced 4 times more O2(-) than the control bacteria, and MT toxicity disappeared in the presence of bathocuproine disulfonic acid, a copper chelator. In contrast, the tetra-domained form did not. Interestingly, in bacteria producing superoxide dismutase but hypersensitive to oxidative stress due to either mutations in thioredoxin and glutathione reductase pathways (WM104 mutant) or to a lack of gamma-glutamylcysteine synthetase (gshA mutant), both chimeric MTs were protecting against Cd toxicity. However, an unexpected lack of antioxidant function was observed for both chimeric MTs, which were found to enhance the toxicity of hydrogen peroxide in WM104, or that of menadione in QC1726. Altogether, our results suggest that superoxide dismutase activity counteracts the potential prooxidative effect of the tri-domained MT mediated by Cu ions and that the tetra-domained form is a very efficient protector against metal toxicity in vivo.     

Aluminium toxicity and binding to Escherichia coli

The toxicity and binding of aluminium to Escherichia coli has been studied. Inhibition of growth by aluminium nitrate was markedly dependent on pH; growth in medium buffered to pH 5.4 was more sensitive to 0.9 mM or 2.25 mM aluminium than was growth at pH 6.6–6.8. In medium buffered with 2-(N-morpholino)ethanesulphonic acid (MES), aluminium toxicity was enhanced by omission of iron from the medium or by use of exponential phase starter cultures. Analysis of bound aluminium by atomic absorption spectroscopy showed that aluminium was bound intracellularly at one type of site with a K _m of 0.4 mM and a capacity of 0.13 mol (g dry wt)^-1. In contrast, binding of aluminium at the cell surface occurred at two or more sites with evidence of cooperativity. Addition of aluminium nitrate to a weakly buffered cell suspension caused acidification of the medium attributable to displacement of protons from cell surfaces by metal cations. It is concluded that aluminium toxicity is related to pH-dependent speciation [with Al(H₂O) ₆ ³⁺ probably being the active species] and chelation of aluminium in the medium. Aluminium transport to intracellular binding sites may involve Fe(III) transport pathways.     

Metal binding to the HIV nucleocapsid peptide

 Co(II) and Zn(II) binding constants have been measured for binding to the HIV-1 nucleocapsid N-terminal metal binding domain (residues 1–18), using competition titration methods and monitoring Co(II) binding by visible absorbance spectroscopy. Enthalpies for binding were directly measured by isothermal titration colorimetry. The results are compared with recent studies of related systems, including a study of Zn(II) binding by the full length protein. Received: 1 December 1998 / Accepted: 31 December 1998