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1.
为了明确重金属Cd2+对拟水狼蛛Pirata subpiraticus体内金属硫蛋白(metallothionein, MT)含量及其生长发育的影响, 在室内条件下用5个不同浓度(0, 10, 20, 40和80 μg/g)的Cd2+培养的黑腹果蝇Drosophila melanogaster饲喂从5种不同生境下(S1, S2, S3, S4和S5)采集的拟水狼蛛性成熟雌蛛产卵孵化的幼蛛,待幼蛛性成熟后取所得雌蛛成蛛采用原子吸收光谱法测定了Cd2+ 诱导下拟水狼蛛体内金属硫蛋白含量及其存活率和生长率。结果表明: 食物中过量的Cd2+能够通过食物链进行传递并在拟水狼蛛体内积累, 积累量随黑腹果蝇培养基中Cd2+浓度的增加而增加,存在显著的浓度-效应关系。不同浓度的Cd2+能够诱导拟水狼蛛体内MT不同的表达,表达量与Cd2+浓度显著正相关(P<0.05)。当浓度低于20 μg/g时,污染点(S1, S2, S3和S4)拟水狼蛛体内MT表达量显著高于参照组S5(P<0.05); 当高于20 μg/g时, 所有样点拟水狼蛛体内MT表达量差异不显著(P>0.05)。拟水狼蛛存活率和成长率随着Cd2+浓度的升高呈下降趋势。据此认为,金属硫蛋白可能是蜘蛛耐受重金属污染的重要机制,与重金属具有一定浓度-效应关系。 相似文献
2.
Kavita Shah Prerna Singh Sareeta Nahakpam 《Journal of plant biochemistry and biotechnology.》2013,22(1):103-112
Excess cadmium (Cd2+) in the soil environment is taken up by plants and can cause phytotoxicity. Elevated temperatures also lead to deleterious effects on plants. Plants are very often exposed to a combination of stresses rather than a single stress. The effect of Cd2+ and heat stress (HS) on the growth, root ultrastructure, lipid peroxidation (MDA), hydrogen peroxide accumulation and the activities of antioxidant enzymes peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) of rice roots from sensitive cv. DR-92 and tolerant cv. Bh-1 were investigated at 10 and 20 day of growth under controlled conditions. At day 10 under all Cd2+ treatments, the Cd2+ content between the two rice cultivars were almost similar. Application of 500 μM Cd2+ significantly increased metal concentrations at day 20 in the roots of rice seedlings resulting in a maximum accumulation of 44.25 μg Cd2+ g-1 dry wt in cv. DR-92 and 30 μg Cd2+ g-1 dry wt in cv. Bh-1 with a ~25 % decline in Relative Growth Index (RGI) in cv. DR-92. TEM studies revealed slight disorganization with cell wall ingrowths in root tissues from cv. DR-92 grown in 100 μM Cd2+ + HS. Uptake and accumulation of Cd2+ increased upon heat treatment in parenchyma, vacuoles and vascular cylinder of root tissues. Peroxidase primarily located in cell walls, the intensity being higher in sensitive cv. DR-92. Under Cd2+ stress alone, plants of sensitive cv. DR-92 significantly increased the H2O2 and MDA levels together with increased activities of the enzymes POD, CAT and APX at day 10 but remained almost stable at day 20. A strong increase in MDA levels was noted at day 20 in tolerant cv. Bh-1. Cd2+ + HS treatments in tolerant cv.Bh-1 led to a decreased H2O2 and MDA levels and decreased activities of the enzymes POD, CAT and APX. Results suggest stimulation of root antioxidant system under combination of two stresses and that heat stress seem to have a direct protective role by mitigating the effect of mild Cd2+ toxicity largely by enhanced Cd2+-MT formation contributing thereby towards the management of Cd2+ toxicity at cellular level that confers Cd2+ tolerance to rice cv. Bh-1. 相似文献
3.
Previous work in our laboratory led to the isolation of a cadmium (Cd)-resistant variant (Cdr2C10) of the line CHO Chinese Hamster cell having a 10-fold greater resistance to the cytotoxic action of Cd2+ compared with the CHO cell. This resistance was attributed to an increased capacity of the Cd2+-resistant Cdr2C10 subline to induce synthesis of the Cd2+- and Zn2+-binding protein(s), metallothionein(s) (MT). Evidence that Cd2+ behaves as an analog of the essential trace metal, Zn2+, especially as an inducer of MT synthesis, suggested that the Cdr and CHO cell types could be employed to investigate cellular Zn2+ metabolism. In the present study, measurements were made to compare CHO and Cdr cell types for (a) growth as a function of the level of ZnCl2 added to the culture medium, (b) uptake and subcellular distribution of Zn2+, and (c) capacity to induce MT synthesis. The results of these measurements indicated that (a) both CHO and Cdr cell types grew normally (T
d≊16–18 h) during exposures to Zn2+ at levels up to 100 μM added to the growth medium, but displayed abrupt growth inhibition at higher Zn2+ levels, (b) Cdr cells incorporate fourfold more Zn2+ during a 24-h exposure to the maximal subtoxic level of Zn2+ and (c) the CHO cell lacks the capacity to induce MT synethesis while the Cdr cell is proficient in this response during exposure to the maximal subtoxic Zn2+ level. These findings suggest that (a) the CHO and Cdr cell systems will be useful in further studies of cellular Zn2+ metabolism, especially in comparisons of Zn2+ metabolism in the presence and absence of induction of the Zn2+-sequestering MT and (b) a relationship exists between cellular capacity to induce MT synthesis and capacity for cellular
Zn2+ uptake. 相似文献
4.
5.
Hong-Xia Lan Zhou-Fei Wang Qi-Hong Wang Mei-Mei Wang Yong-Mei Bao Ji Huang Hong-Sheng Zhang 《Molecular biology reports》2013,40(2):1201-1210
The CDF family is a ubiquitous family that has been identified in prokaryotes, eukaryotes, and archaea. Members of this family are important heavy metal transporters that transport metal ions out of the cytoplasm. In this research, a full length cDNA named Oryza sativa Zn Transporter 1 (OZT1) that closely related to rat ZnT-2 (Zn Transporter 2) gene was isolated from rice. The OZT1 encoding a CDF family protein shares 28.2 % ~ 84.3 % of identities and 49.3 % ~ 90.9 % of similarities with other zinc transporters such as RnZnT-2, HsZnT-8, RnZnT-8 and AtMTP1. OZT1 was constitutively expressed in various rice tissues. The OZT1 expression was significantly induced both in the seedlings of japonica rice Nipponbare and indica rice IR26 in response to Zn2+ and Cd2+ treatments. Besides, OZT1 expression was also increased when exposed to other excess metals, such as Cu2+, Fe2+ and Mg2+. Subcellular localization analysis indicated that OZT1 localized to vacuole. Heterologous expression of OZT1 in yeast increased tolerance to Zn2+ and Cd2+ stress but not the Mg2+ stress. Together, OZT1 is a CDF family vacuolar zinc transporter conferring tolerance to Zn2+ and Cd2+ stress, which is important to transporting and homeostasis of Zn, Cd or other heavy metals in plants. 相似文献
6.
7.
Metallothioneins (MTs) are ubiquitous, low molecular mass and cysteine-rich proteins that play important roles in maintaining intracellular metal homeostasis, eliminating metal toxification and protecting the cells against oxidative damages. MTs are able to bind metal ions through the thiol groups of their cysteine residues. Plants have several MT isoforms which are classified into four types based on the arrangement of cysteine residues. In the present study, a rice (Oryza sativa) gene encoding type 1 MT isoform, OsMTI-1b, was inserted in vector pET41a and overexpressed in Escherichia coli as carboxy-terminal extensions of glutathione-S-transferase (GST). The recombinant protein GST-OsMTI-1b was purified using affinity chromatography and its ability to bind with Ni2+, Cd2+, Zn2+ and Cu2+ ions was analyzed. The results demonstrated that this isoform has ability to bind Ni2+, Cd2+ and Zn2+ ions in vitro, whereas it has no substantial ability to bind Cu2+ ions. From competitive reaction with 5,5′-dithiobis(2-nitrobenzoic acid), DTNB, the affinity of metal ions for recombinant form of GST-OsMTI-1b was as follows: Ni2+/Cd2+ > Zn2+ > Cu2+ 相似文献
8.
Basharat Ali Rafaqat A. Gill Su Yang Muhammad B. Gill Muhammad A. Farooq Dan Liu Muhammad K. Daud Shafaqat Ali Weijun Zhou 《PloS one》2015,10(4)
It is evident from previous reports that 5-aminolevulinic acid (ALA), like other known plant growth regulators, is effective in countering the injurious effects of heavy metal-stress in oilseed rape (Brassica napus L.). The present study was carried out to explore the capability of ALA to improve cadmium (Cd2+) tolerance in B. napus through physiological, molecular, and proteomic analytical approaches. Results showed that application of ALA helped the plants to adjust Cd2+-induced metabolic and photosynthetic fluorescence changes in the leaves of B. napus under Cd2+ stress. The data revealed that ALA treatment enhanced the gene expressions of antioxidant enzyme activities substantially and could increase the expression to a certain degree under Cd2+ stress conditions. In the present study, 34 protein spots were identified that differentially regulated due to Cd2+ and/or ALA treatments. Among them, 18 proteins were significantly regulated by ALA, including the proteins associated with stress related, carbohydrate metabolism, catalysis, dehydration of damaged protein, CO2 assimilation/photosynthesis and protein synthesis/regulation. From these 18 ALA-regulated proteins, 12 proteins were significantly down-regulated and 6 proteins were up-regulated. Interestingly, it was observed that ALA-induced the up-regulation of dihydrolipoyl dehydrogenase, light harvesting complex photo-system II subunit 6 and 30S ribosomal proteins in the presence of Cd2+ stress. In addition, it was also observed that ALA-induced the down-regulation in thioredoxin-like protein, 2, 3-bisphosphoglycerate, proteasome and thiamine thiazole synthase proteins under Cd2+ stress. Taken together, the present study sheds light on molecular mechanisms involved in ALA-induced Cd2+ tolerance in B. napus leaves and suggests a more active involvement of ALA in plant physiological processes than previously proposed. 相似文献
9.
Stefania Cigliano Paolo Remondelli Liliana Minichiello Maria Cristina Mellone Gianluca Martire Stefano Bonatti Arturo Leone 《Experimental cell research》1996,228(2):173
The expression of metallothionein (MT) and heat shock protein gene families was investigated in normal and in HeLa-derived cadmium-resistant cells, named H454. In the absence of amplification of MT genes H454 cells accumulated elevated concentrations of cadmium ions and synthesized higher levels of MT proteins than unselected HeLa cells. Northern blot analyses revealed higher levels of MT mRNAs in the resistant cells than in wild-type cells after Cd2+and Zn2+exposure. Evaluation of the cytotoxic potential of the different metals confirmed the high resistance to cadmium of the H454 cells. Two proteins of the heat shock family, hsp70 and GRP78, were synthesized in Cd2+-exposed H454 cells at levels comparable to the ones present in Cd2+-treated normal cells. Northern blot analyses of the mRNA levels corresponding to these proteins revealed elevated expression of both hsp70 and GRP78 mRNAs in H454 cells upon exposure to cadmium ions and no response to zinc induction. These data suggest the existence in the H454 cells of a cadmium-specific pathway of regulation of MT and heat shock genes. 相似文献
10.
Cellular and Subcellular Immunohistochemical Localization and Quantification of Cadmium Ions in Wheat (Triticum aestivum) 总被引:1,自引:0,他引:1
Wei Gao Tiegui Nan Guiyu Tan Hongwei Zhao Weiming Tan Fanyun Meng Zhaohu Li Qing X. Li Baomin Wang 《PloS one》2015,10(5)
The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC) imaging method for cadmium ions (Cd2+) was developed for the first time for the wheat Triticum aestivum grown in Cd2+-fortified soils. Also, 1-(4-Isothiocyanobenzyl)-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA) was used to chelate the mobile Cd2+. The ITCB-EDTA/Cd2+ complex was fixed with proteins in situ via the isothiocyano group. A new Cd2+-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd2+-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd2+-positive roots were compared with those of Cd2+-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd2+ content in plant tissues and illustrate Cd2+ translocation and cellular and subcellular responses of T. aestivum to Cd2+ stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd2+ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd2+ in plant tissues and can be extended to other heavy metallic ions. 相似文献
11.
Lone B?kgaard Maria D. Mikkelsen Danny M. S?rensen Josefine N. Hegelund Daniel P. Persson Rebecca F. Mills Zhang Yang S?ren Husted Jens Peter Andersen Morten J. Buch-Pedersen Jan K. Schjoerring Lorraine E. Williams Michael G. Palmgren 《The Journal of biological chemistry》2010,285(41):31243-31252
Heavy metal pumps (P1B-ATPases) are important for cellular heavy metal homeostasis. AtHMA4, an Arabidopsis thaliana heavy metal pump of importance for plant Zn2+ nutrition, has an extended C-terminal domain containing 13 cysteine pairs and a terminal stretch of 11 histidines. Using a novel size-exclusion chromatography, inductively coupled plasma mass spectrometry approach we report that the C-terminal domain of AtHMA4 is a high affinity Zn2+ and Cd2+ chelator with capacity to bind 10 Zn2+ ions per C terminus. When AtHMA4 is expressed in a Zn2+-sensitive zrc1 cot1 yeast strain, sequential removal of the histidine stretch and the cysteine pairs confers a gradual increase in Zn2+ and Cd2+ tolerance and lowered Zn2+ and Cd2+ content of transformed yeast cells. We conclude that the C-terminal domain of AtHMA4 serves a dual role as Zn2+ and Cd2+ chelator (sensor) and as a regulator of the efficiency of Zn2+ and Cd2+ export. The identification of a post-translational handle on Zn2+ and Cd2+ transport efficiency opens new perspectives for regulation of Zn2+ nutrition and tolerance in eukaryotes. 相似文献
12.
Urszula Guzik Katarzyna Hupert-Kocurek Karina Sałek Danuta Wojcieszyńska 《World journal of microbiology & biotechnology》2013,29(2):267-273
The aim of this paper was to describe the effect of various metal ions on the activity of protocatechuate 3,4-dioxygenase from Stenotrophomonas maltophilia KB2. We also compared activity of different dioxygenases isolated from this strain, in the presence of metal ions, after induction by various aromatic compounds. S. maltophilia KB2 degraded 13 mM 3,4-dihydroxybenzoate, 10 mM benzoic acid and 12 mM phenol within 24 h of incubation. In the presence of dihydroxybenzoate and benzoate, the activity of protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase was observed. Although Fe3+, Cu2+, Zn2+, Co2+, Al3+, Cd2+, Ni2+ and Mn2+ ions caused 20–80 % inhibition of protocatechuate 3,4-dioxygenase activity, the above-mentioned metal ions (with the exception of Ni2+) inhibited catechol 1,2-dioxygenase to a lesser extent or even activate the enzyme. Retaining activity of at least one of three dioxygenases from strain KB2 in the presence of metal ions makes it an ideal bacterium for bioremediation of contaminated areas. 相似文献
13.
《Bioscience, biotechnology, and biochemistry》2013,77(12):2650-2656
An acidophilic volvocine flagellate, Chlamydomonas acidophila (Volvocales) that was isolated from an acid lake, Katanuma, in Miyagi prefecture, Japan was studied for growth, ultrastructural characterization, and metal tolerance.Chlamydomonas acidophila is obligately photoautotrophic, and did not grow in the cultures containing acetate or citrate even in the light. The optimum pH for growth was 3.5-4.5. To characterize metal tolerance, the toxic effects of Cd, Co, Cu, and Zn on this alga were also studied. Effective metal concentrations, which limited the growth by 50%, EC50 were measured, after 72h of static exposure. EC50s were 14.4 μM Cd2+, 81.3 μM Co2+, 141μM Cu2+, and 1.16 mM Zn2+ for 72 h of exposure. Thus, this alga had stronger tolerance to these metals than other species in the genus Chlamydomonas. 相似文献
14.
Qing Hu Hongyan Qi Zhihui Bai Minna Dou Jinghai Zeng Fanyuanhang Zhang Hongxun Zhang 《World journal of microbiology & biotechnology》2007,23(7):971-976
A Cd2+-hyperresistant bacterial strain HQ-1 was isolated from a lead–zinc mine. The strain was characterized and identified as Bacillus cereus based on morphology, physiological tests and 16S rRNA gene analysis. The minimal inhibitory concentration of Cd2+ for the bacterium was 0.012 mol/l. Isotherms for cadmium (Cd) biosorption by cells of B. cereus strain HQ-1 were investigated. The equilibrium data could be fitted by a Langmuir isotherm equation. The possible functional
sites that might be influenced by the sorption were determined. The results indicate that this B. cereus strain has excellent potential for biosorption of Cd. Physiological characterization of the isolate also indicates possible
application of this strain for bioremediation of sites with Cd contamination. 相似文献
15.
Anna Espart Maribel Marín Selene Gil-Moreno òscar Palacios Francisco Amaro Ana Martín-González Juan C. Gutiérrez Mercè Capdevila Sílvia Atrian 《International journal of biological sciences》2015,11(4):456-471
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). 相似文献
16.
In vitro selection of RNA-cleaving DNAzymes is a powerful method for isolating metal-specific DNA. A few successful examples are known, but it is still difficult to target some thiophilic metals such as Cd2+ due to limited functional groups in DNA. While using modified bases expands the chemical functionality of DNA, a single phosphorothioate modification might boost its affinity for thiophilic metals without complicating the selection process or using bases that are not commercially available. In this work, the first such in vitro selection for Cd2+ is reported. After using a blocking DNA and negative selections to rationally direct the library outcome, a highly specific DNAzyme with only 12 nucleotides in the catalytic loop is isolated. This DNAzyme has a cleavage rate of 0.12 min−1 with 10 μM Cd2+ at pH 6.0. The Rp form of the substrate is cleaved ∼100-fold faster than the Sp form. The DNAzyme is most active with Cd2+ and its selectivity against Zn2+ is over 100 000-fold. Its application in detecting Cd2+ is also demonstrated. The idea of introducing single modifications in the fixed region expands the scope of DNA/metal interactions with minimal perturbation of DNA structure and property. 相似文献
17.
Annie Y.C. Law Martin J. Stillman 《Biochemical and biophysical research communications》1984,121(3):1006-1013
The detailed spectral changes observed in the absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra upon addition of Cd2+ to rat liver Cd, Zn-metallothionein (MT) are reported. Results from dialysis experiments clearly demonstrate that up to 8.6 mole equivalents of Cd2+ can be bound to this protein. The excess Cd2+ ions bound appear to have lower binding constants than those of the first seven Cd2+ ions bound. Red blood cell hemolysate (RBC) can compete with the metallothionein for all Cd2+ bound in excess of seven mole equivalents. Thus the RBC hemolysate method of estimating protein concentrations is shown to be correct when based upon complete loading of all binding sites in MT with Cd2+. 相似文献
18.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1988,952(2):222-229
Homogeneous preparations of l-threonine dehydrogenase (l-threonine: NAD+ oxidoreductase, EC 1.1.1.103) from Escherichia coli K-12, after having been dialyzed against buffers containing Chelex-100 resin, have a basal level of activity of 10–20 units/mg. Added Cd2+ stimulates dehydrogenase activity approx. 10-fold; this activation is concentration-dependent and is saturable with an activation Kd = 0.9 μM. Full activation by Cd2+ is obtained in the absence of added thiols. The pH-activity profile of the Cd2+-activated enzyme conforms to a theoretical curve for one-proton ionization with a pKa = 7.85. Mn2+, the only other activating metal ion, competes with Cd2+ for the same binding site. Km values forl-threonine and NAD+ as well as the Vmax for ‘demetallized’, Cd2+-activated, and Mn2+-activated threonine dehydrogenase were determined and compared. 相似文献
19.
Jennifer Abbruzzese Frank B. Sachse Martin Tristani-Firouzi Michael C. Sanguinetti 《The Journal of general physiology》2010,136(2):203-224
Each of the four subunits in a voltage-gated potassium channel has a voltage sensor domain (VSD) that is formed by four transmembrane helical segments (S1–S4). In response to changes in membrane potential, intramembrane displacement of basic residues in S4 produces a gating current. As S4 moves through the membrane, its basic residues also form sequential electrostatic interactions with acidic residues in immobile regions of the S2 and S3 segments. Transition metal cations interact with these same acidic residues and modify channel gating. In human ether-á-go-go–related gene type 1 (hERG1) channels, Cd2+ coordinated by D456 and D460 in S2 and D509 in S3 induces a positive shift in the voltage dependence of activation of ionic currents. Here, we characterize the effects of Cd2+ on hERG1 gating currents in Xenopus oocytes using the cut-open Vaseline gap technique. Cd2+ shifted the half-point (V1/2) for the voltage dependence of the OFF gating charge–voltage (QOFF-V) relationship with an EC50 of 171 µM; at 0.3 mM, V1/2 was shifted by +50 mV. Cd2+ also induced an as of yet unrecognized small outward current (ICd-out) upon repolarization in a concentration- and voltage-dependent manner. We propose that Cd2+ and Arg residues in the S4 segment compete for interaction with acidic residues in S2 and S3 segments, and that the initial inward movement of S4 associated with membrane repolarization displaces Cd2+ in an outward direction to produce ICd-out. Co2+, Zn2+, and La3+ at concentrations that caused ∼+35-mV shifts in the QOFF-V relationship did not induce a current similar to ICd-out, suggesting that the binding site for these cations or their competition with basic residues in S4 differs from Cd2+. New Markov models of hERG1 channels were developed that describe gating currents as a noncooperative two-phase process of the VSD and can account for changes in these currents caused by extracellular Cd2+. 相似文献
20.
Duncan E.K. SutherlandKelly L. Summers Martin J. Stillman 《Biochemical and biophysical research communications》2012,426(4):601-607
Mammalian metallothioneins (MTs) are a family of small cysteine rich proteins believed to have a number of physiological functions, including both metal ion homeostasis and toxic metal detoxification. Mammalian MTs bind 7 Zn2+ or Cd2+ ions into two distinct domains: an N-terminal β-domain that binds 3 Zn2+ or Cd2+, and a C-terminal α-domain that binds 4 Zn2+ or Cd2+. Although stepwise metalation to the saturated M7-MT (where M = Zn2+ or Cd2+) species would be expected to take place via a noncooperative mechanism involving the 20 cysteine thiolate ligands, literature reports suggest a cooperative mechanism involving cluster formation prior to saturation of the protein. Electrospray ionization mass spectrometry (ESI-MS) provides this sensitivity through delineation of all species (Mn-MT, n = 0-7) coexisting at each step in the metalation process. We report modeled ESI-mass spectral data for the stepwise metalation of human recombinant MT 1a (rhMT) and its two isolated fractions for three mechanistic conditions: cooperative (where the binding affinities are: K1 < K2 < K3 < ··· < K7), weakly cooperative (where K1 = K2 = K3 = ··· = K7), and noncooperative, (where K1 > K2 > K3 > ··· > K7). Detailed ESI-MS metalation data of human recombinant MT 1a by Zn2+ and Cd2+ are also reported. Comparison of the experimental data with the predicted mass spectral data provides conclusive evidence that metalation occurs in a noncooperative fashion for Zn2+ and Cd2+ binding to rhMT 1a. 相似文献