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Staphylococcus aureus pI258 CadC is an extrachromosomally encoded metalloregulatory repressor protein from the ArsR superfamily which negatively regulates the expression of the cad operon in a metal-dependent fashion. The metalloregulatory hypothesis holds that direct binding of thiophilic divalent cations including Cd(II), Pb(II), and Zn(II) by CadC allosterically regulates the DNA binding activity of CadC to the cad operator/promoter (O/P). This report presents a detailed characterization of the metal binding and DNA binding properties of wild-type CadC. The results of analytical ultracentrifugation experiments suggest that both apo- and Cd(1)-CadC are stable or weakly dissociable homodimers characterized by a K(dimer) = 3.0 x 10(6) M(-1) (pH 7.0, 0.20 M NaCl, 25.0 degrees C) with little detectable effect of Cd(II) on the dimerization equilibrium. As determined by optical spectroscopy, the stoichiometry of Cd(II) and Pb(II) binding is approximately 0.7-0.8 mol/mol of wild-type CadC monomer. Chelator (EDTA) competition binding isotherms reveal that Cd(II) binds very tightly, with K(Cd) = 4.3 (+/-1.8) x 10(12) M(-1). The results of UV-Vis and X-ray absorption spectroscopy of the Cd(1) complex are consistent with a tetrathiolate (S(4)) complex formed by four cysteine ligands. The (113)Cd NMR spectrum reveals a single resonance of delta = 622 ppm, consistent with an S(3)(N,O) or unusual upfield-shifted S(4) complex. The Pb(II) complex reveals two prominent absorption bands at 350 nm (epsilon = 4000 M(-1) cm(-1)) and 250 nm (epsilon = 41 000 M(-1) cm(-1)), spectral properties consistent with three or four thiolate ligands to the Pb(II) ion. The change in the anisotropy of a fluorescein-labeled oligonucleotide containing the cad O/P upon binding CadC and analyzed using a dissociable CadC dimer binding model reveals that apo-CadC forms a high-affinity complex [K(a) = (1.1 +/- 0.3) x 10(9) M(-1); pH 7.0, 0.40 M NaCl, 25 degrees C], the affinity of which is reduced approximately 300-fold upon the binding of a single molar equivalent of Cd(II) or Pb(II). The implications of these findings on the mechanism of metalloregulation are discussed. 相似文献
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The Staphylococcus aureus plasmid pI258 CadC is a homodimeric repressor that binds Cd(II), Pb(II), and Zn(II) and regulates expression of the cadAC operon. CadC binds two Cd(II) ions per dimer, with a tetrathiolate binding site composed of residues Cys(7), Cys(11), Cys(58), and Cys(60). It is not known whether each site consists of residues from a single monomer or from residues contributed by both subunits. To examine whether Cys(7) and Cys(11) are spatially proximate to Cys(58) and Cys(60) of the same subunit or of the other subunit, homodimers with the same cysteine mutation in each subunit and heterodimers containing different cysteine mutations in the two subunits were reacted with 4,6-bis(bromomethyl)-3,7-dimethyl-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione, which cross-links thiol groups that are within 3-6 A of each other. Cys(7) or Cys(11) cross-linked only with Cys(58) or Cys(60) on the other subunit. The data demonstrate that Cys(7) and Cys(11) from one monomer are within 3-6 A of either Cys(58) or Cys(60) in the other monomer. The results of this study strongly indicate that each of the two Cd(II) binding sites in the CadC homodimer is composed of Cys(7) and Cys(11) from one monomer and Cys(58) and Cys(60) from the other monomer. 相似文献
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Laura S. Busenlehner Julius L. Apuy David P. Giedroc 《Journal of biological inorganic chemistry》2002,7(4-5):551-559
Staphylococcus aureus pI258 CadC is a metal sensor protein that regulates the expression of the cad operon which encodes metal ion resistance proteins involved in the efficient efflux of Cd(II), Pb(II), Zn(II) and, according to one report, Bi(III) ions. In this paper, direct evidence is presented that Bi(III) binds to CadC and negatively regulates cad operator/promoter (O/P) binding. Optical absorption spectroscopy reveals that dimeric CadC binds approximately 0.8 mol equivalents of Bi(III) per CadC monomer to form a coordination complex characterized by three S(-)-->Bi(III) ligand-to-metal charge transfer transitions, with the longest wavelength absorption band centered at 415 nm (epsilon(415)=4000 M(Bi)(-1) cm(-1)). UV-Vis absorption spectra of wild-type and mutant Cys-->Gly (Ser) substitution CadC mutants compared to [Bi(DTT)(2)], [Bi(GSH)(3)] and [Bi(NAC)](3) model complexes reveal that Cys7, Cys11, Cys60 and Cys58 directly coordinate Bi(III) in a tetrathiolate coordination complex. The apparent affinity derived from a Bi(III)-displacement optical titration with Cd(II) is estimated to be K(Bi)< or =10(12) M(-1). Apo-CadC binds with high affinity [ K(a)=1.1(+/-0.3)x10(9) M(-1); 0.40 M NaCl, pH 7.0, 25 degrees C] to a 5'-fluorescein-labeled cad O/P oligonucleotide,while the binding of one molar equivalent of Bi(III) per CadC monomer (Bi(1)-CadC) reduces the affinity by approximately 170-fold. Strikingly, Bi(III)-responsive negative regulation of cad O/P binding is abrogated for Bi(1)-C60G CadC and severely disrupted in Bi(1)-C7G CadC, whose relative affinity is reduced only 10-fold. The mechanism of Bi(III)-responsive metalloregulation is discussed, based on the findings presented here. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0336-9. 相似文献
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Feeding bioassay results established that the soybean cysteine proteinase inhibitor N (soyacystatin N, scN) substantially inhibits growth and development of western corn rootworm (WCR), by attenuating digestive proteolysis [Zhao, Y. et al. (1996) Plant Physiol. 111, 1299-1306]. Recombinant scN was more inhibitory than the potent and broad specificity cysteine proteinase inhibitor E-64. WCR digestive proteolytic activity was separated by mildly denaturing SDS-PAGE into two fractions and in-gel assays confirmed that the proteinase activities of each were largely scN-sensitive. Since binding affinity to the target proteinase [Koiwa, H. et al. (1998) Plant J. 14, 371-380] governs the effectiveness of scN as a proteinase inhibitor and an insecticide, five peptides (28-33 kDa) were isolated from WCR gut extracts by scN affinity chromatographic separation. Analysis of the N-terminal sequence of these peptides revealed similarity to a cathepsin L-like cysteine proteinase (DvCAL1, Diabrotica virgifera virgifera cathepsin L) encoded by a WCR cDNA. Our results indicate that cathepsin L orthologs are pivotal digestive proteinases of WCR larvae, and are targets of plant defensive cystatins (phytocystatins), like scN. 相似文献
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The acid-base and coordination properties towards Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II) of four polyamino-phenol macrocycles 15-hydroxy-3,6,9-triazabicyclo[9.3.1]pentadeca-11,13,115-triene L1, 18-hydroxy-3,6,9,12-tetraazabicyclo[12.3.1]octadeca-14,16,118-triene L2, 21-hydroxy-3,6,9,12,15-pentaazabicyclo[15.3.1]enaicosa-17,19,121-triene L3 and 24-hydroxy-3,6,9,12,15,18-hexaazabicyclo[18.3.1]tetraicosa-20,22,124-triene L4 are reported. The protonation and stability constants were determined by means of potentiometric measurements in 0.15 mol dm−3 NMe4Cl aqueous solution at 298.1 K. L1 forms highly unsaturated Co(II), Cu(II), Zn(II) and Cd(II) mononuclear complexes that are prone to give dimeric dinuclear species with [(MH−1L1)2]2+ stoichiometry, in solution. L2 forms stable Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II) mononuclear complexes that can coordinate external species as OH− anion, giving hydroxylated complexes at alkaline pH. L3 forms stable Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II) mononuclear complexes and Co(II), Ni(II), Cu(II) and Zn(II) dinuclear [M2H−1L3]3+ species. L4 forms stable mono- and dinuclear Co(II), Cu(II), Zn(II) and Cd(II) complexes, but only mononuclear species with Pb(II). The effect of macrocyclic size is considered in the discussion of results. 相似文献
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Choi DW Do YS Zea CJ McEllistrem MT Lee SW Semrau JD Pohl NL Kisting CJ Scardino LL Hartsel SC Boyd ES Geesey GG Riedel TP Shafe PH Kranski KA Tritsch JR Antholine WE DiSpirito AA 《Journal of inorganic biochemistry》2006,100(12):2150-2161
Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ. 相似文献
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Yahui Wang Liming Li Xuegang Zou Ranjun Shu Ling Ding Kun Yao 《Soil & Sediment Contamination》2016,25(6):700-715
In situ immobilization constitutes a promising technology for the mitigation of contaminants, through the reduction of metal bioavailability and mobility. This study investigated the adsorption isotherms and kinetic characteristics of humin extracted from peat soils. We also studied the influences of the pH, ionic strengths, and soluble organic matter concentrations of soil solutions on the adsorptive properties of humin, and compared its ability to detoxify potentially toxic metals in both actual and simulated soil solutions. The study results indicated that humin contains a massive population of oxygen-containing functional groups. Its adsorption capacity for Pb(II) was greater than that for Cu(II), which exceeded that for Cd(II). The adsorption of humin for Pb(II) conformed to the Freundlich model, while the adsorption of humin for Cd(II) and Cu(II) followed the Langmuir model. The adsorption kinetics of humin with respect to potentially toxic metals aligned well with second-order kinetics equations. As the pH was elevated, the potentially toxic metal adsorption by humin increased rapidly. Electrolyte ions and tartaric acids in solution both inhibited the adsorption of potentially toxic metals by humin, and its ability to inactivate potentially toxic metals. This was shown to be improved in actual field soil solutions in contrast to simulated soil solutions. 相似文献
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Dong W. Choi Young S. Do Corbin J. Zea Marcus T. McEllistrem Sung-W. Lee Jeremy D. Semrau Nicola L. Pohl Clint J. Kisting Lori L. Scardino Scott C. Hartsel Eric S. Boyd Gill G. Geesey Theran P. Riedel Peter H. Shafe Kim A. Kranski John R. Tritsch William E. Antholine Alan A. DiSpirito 《Journal of inorganic biochemistry》2006,100(12):2150
Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ. 相似文献
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Arsenic efflux governed by the arsenic resistance determinant of Staphylococcus aureus plasmid pI258. 总被引:4,自引:3,他引:4
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The arsenic resistance operon of Staphylococcus aureus plasmid pI258 determined lowered net cellular uptake of 73As by an active efflux mechanism. Arsenite was exported from the cells; intracellular arsenate was first reduced to arsenite and then transported out of the cells. Resistant cells showed lower accumulation of 73As originating from both arsenate and arsenite. Active efflux from cells loaded with arsenite required the presence of the plasmid-determined arsB gene. Efflux of arsenic originating as arsenate required the presence of the arsC gene and occurred more rapidly with the addition of arsB. Inhibitor studies with S. aureus loaded with arsenite showed that arsenite efflux was energy dependent and appeared to be driven by the membrane potential. With cells loaded with 73AsO4(3-), a requirement for ATP for energy was observed, leading to the conclusion that ATP was required for arsenate reduction. When the staphylococcal arsenic resistance determinant was cloned into Escherichia coli, lowered accumulation of arsenate and arsenite and 73As efflux from cells loaded with arsenate were also found. Cloning of the E. coli plasmid R773 arsA gene (the determinant of the arsenite-dependent ATPase) in trans to the S. aureus gene arsB resulted in increased resistance to arsenite. 相似文献
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This study describes the preparation of two new chelating materials derived from succinylated mercerized cellulose (cell 1). Cell 1 was activated through two different methods by using diisopropylcarbodiimide and acetic anhydride (to form an internal anhydride) and reacted with triethylenetetramine in order to obtain cell 2 and 4. New modified celluloses were characterized by mass percent gain, concentration of amine functions, elemental analysis, and infrared spectroscopy. Cell 2 and 4 showed degrees of amination of 2.8 and 2.3 mmol/g and nitrogen content of 6.07% and 4.61%, respectively. The capacity of cell 2 and 4 to adsorb Cu2+, Cd2+, and Pb2+ ions from single aqueous solutions were examined. The effect of contact time, pH, and initial concentration of metal ions on the metal ions uptake was also investigated. Adsorption isotherms were well fitted by the Langmuir model. The maximum adsorption capacity of cell 2 and 4 were found to be 56.8 and 69.4 mg/g for Cu2+; 68.0 and 87.0 mg/g for Cd2+; and 147.1 and 192.3 mg/g for Pb2+, respectively. 相似文献
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Tsai KJ Lin YF Wong MD Yang HH Fu HL Rosen BP 《Journal of bioenergetics and biomembranes》2002,34(3):147-156
Plasmid pl258 carries the cadA gene that confers resistance to cadmium, lead, and zinc. CadA catalyzes ATP-dependent cadmium efflux from cells of Staphylococcus aureus. It is a member of the superfamily of P-type ATPases and belongs to the subfamily of soft metal ion pumps. In this study the membrane topology of this P-type ATPase was determined by constructing fusions with the topological reporter genes phoA or lacZ. A series of 44 C-terminal truncated CadAs were fused with one or the other reporter gene, and the activity of each chimeric protein was determined. In addition, the location of the first transmembrane segment was determined by immunoblot analysis. The results are consistent with the pl258 CadA ATPase having eight transmembrane segments. The first 109 residues is a cytosolic domain that includes the Cys(X)2Cys motif that distinguishes soft metal ion-translocating P-type ATPases from their hard metal ion-translocating homologues. Another feature of soft metal ion P-type ATPases is the CysProCys motif, which is found in the sixth transmembrane segment of CadA. The phosphorylation site and ATP binding domain conserved in all P-type ATPases are situated within the large cytoplasmic loop between the sixth and seventh transmembrane segments. 相似文献
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《Chemical Speciation and Bioavailability》2013,25(4):231-239
AbstractComplexation of toxic metal ions with maleic acid in (0.0–2.5% w/v) cetyltrimethylammonium bromide (CTAB)–water mixtures has been studied pH-metrically at ambient conditions and an ionic strength of 0.16 mol L-1. The existence of different binary species was established from modelling studies using the computer program MINIQUAD75. The best-fit chemical models were selected based on statistical parameters such as the crystallographic R factor and sum of the squares of residuals in mass-balance equations. The models for binary complex systems contain the chemical species ML2, ML2H and ML3 for Pb(II), Cd(II) and Hg(II) in CTAB–water mixtures. The trend in the variation of stability constants with change in the mole fraction of the medium was explained based on electrostatic and non-electrostatic forces. Distribution of the species with pH at different compositions of CTAB–water mixtures was also presented. 相似文献
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Zn(II)-113Cd(II) and Zn(II)-Mg(II) hybrids of alkaline phosphatase. 31P and 113Cd NMR 总被引:1,自引:0,他引:1
Methods have been developed for the addition of different metal ion species to the three distinct pairs of metal sites (A, B, and C) found in the dimer of apoalkaline phosphatase. This allows the preparation of hybrid alkaline phosphatases in which A and B sites of each monomer contain two different species of metal ion or the A and B sites of one monomer contain the same species of metal ion, while the adjacent monomer contains a second species. The following hybrids have been characterized in detail: (Zn(II)ACd(II)B)2 alkaline phosphatase, (Zn(II)AMg(II)B)2 alkaline phosphatase, (Cd(II)AZn(II)B)2 alkaline phosphatase, and (Zn(II)AZn(II]B)(Cd(II)ACd(II)B) alkaline phosphatase. 31P and, where appropriate, 113Cd NMR have been used to monitor the behavior of the covalent (E-P) and noncovalent (E X P) phosphointermediates and of the A and B metal ions. From the pH dependencies of the E-P in equilibrium E X P in equilibrium E + Pi equilibria, it is clear that A site metal is the dominant influence in dephosphorylation of E-P and may have a coordinated water molecule, which ionizes to ZnOH- at a low pH providing the nucleophile for dephosphorylation. A site metal also serves to coordinate phosphate in the E X P complex. B site metal has a much smaller effect on dephosphorylation rates, although it does dramatically alter the Pi dissociation rate, which is the rate-limiting step for the native enzyme at alkaline pH, and is probably important in neutralizing the charge on the phosphoseryl residue, thus potentiating the nucleophilic attack of the OH- bound at A site. Phosphate dissociation is slowed markedly by replacement of B site zinc by cadmium. There is clear evidence for long range effects of subunit-subunit interactions, since metal ion and phosphate binding at one active center alters the environments of A and B site metal ions and phosphoserine at the other active site. 相似文献
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生长在铅锌矿废水污灌区的长豇豆组织中Pb、Zn、Cd含量的品种间差异 总被引:1,自引:1,他引:1
在经铅锌矿废水灌溉约50年的农田中种植24个长豇豆品种,测定植物体内的Pb、Zn、Cd在根、茎、叶及果实中的含量。结果表明,Cd在长豇豆根、茎、叶及果实的平均含量分别为1.212、0.425、1.051mg·kg^-1和0.011mg·kg^-1;Pb则分别是92.53、9.79、33.08mg·kg^-1和0.120mg·kg^-1;Zn分别是130.14、59.40、99.94mg·kg^-1和6.320mg·kg^-1。果实中的Cd、Pb和Zn最大品种间差异分别达4.4倍、4.2倍和1.6倍;各品种3种重金属含量ANOVA分析结果显示了品种间差异具有极显著意义。不同仁色(花仁、红仁及黑仁)长豇豆品种间的3种重金属含量在根部均有显著差异,而Zn及Cd含量在茎组织中也有显著差异;但各组在叶及果实中没有显著差异。尽管污灌区土壤Cd、Pb和Zn浓度均超出了国家土壤环境质量标准二级土壤的最高限值,但绝大多数品种的果实中所含重金属均符合国家食品卫生标准。Pb较易在果实中积累,有一个品种果实Pb浓度超过国家标准。根和茎中的3种重金属含量相互间均具有高度相关性,且果实中的Cd和Pb含量间也有显著相关,表明长豇豆对Cd、Pb和Zn的吸收和积累有协同性,这一特性使得同时低量积累重金属的长豇豆品种的筛选更为容易,特别是在可食部分同时低量积累Cd和Pb的品种。污灌区具有比对照区更高的产量,说明长豇豆能耐受农田中Cd、Pb和Zn的复合污染,因而生产者比较难以从长豇豆的中毒症状发现重金属的污染,导致在污染土壤中生产长豇豆容易受重金属污染。可见,筛选和培育低量积累重金属的长豇豆品种有利于降低人类通过食物链暴露于重金属的水平。 相似文献